Lapres Protein.docx

I.

Title of Experiment

: Understanding Characteristic and Color Reaction of Protein

II. Starting Experiment Finishing Experiment III. The Aim of Experiment

: Tuesday / April, 30th 2018, 10.20 AM : Tuesday / April, 03th 2018, 16.20 PM :

1. Distinguish the solubility of protein in a reversible and irreversible way. 2. Distinguish protein denaturation reaction caused acid, salt, salt from heavy metal, as well as heated based on observation. 3. Understanding the causes of precipitate of protein. 4. Identify the presence of protein through a color reaction. IV. Basic Theory A. Amino Acid Amino acids are carboxylic acids that contain an amine function. Under certain conditions the amine group of one molecule and the carboxyl group of a second can react, uniting the two amino acids by an amide bond.

Picture 1. Amino Acid Carey, 2000. Amino acids are classified as , ,  and so on, according to the location of the amine group on the carbon chain that contains the carboxylic acid function.

Picture 2. The example from Classification of amino acid Carey, 2000. B. Acid Base Behavior of Amino Acid The carboxyl group is deprotonated and exists as the carboxylate anion at a physiological pH of 7.3, while an amino group is protonated and exists as the

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1

ammonium cation. Thus, amino acids exist in aqueous solution primarily in the form of a dipolar ion, or zwitterion (German zwitter, meaning “hybrid”). Amino acid zwitterions are internal salts and therefore have many of the physical properties associated with salts. They have large dipole moments, are soluble in water but insoluble in hydrocarbons, and are crystalline substances with relatively high melting points. In addition, amino acids are amphiprotic; they can react either as acids or as bases, depending on the circumstances. In aqueous acid solution, an amino acid zwitterion is a base that accepts a proton to yield a cation; in aqueous base solution, the zwitterion is an acid that loses a proton to form an anion. Note that it is the carboxylate, CO2, that acts as the basic site and accepts a proton in acid solution, and it is the ammonium cation, NH3. That acts as the acidic site and donates a proton in base solution McMurry, 2008.

Picture 3. Structure in acid and base solution McMurry, 2008. The dipolar nature of amino acid gives them some unusual properties 1. Amino acid have high melting points, generally over 200C. 2. Amino acid are more soluble in water than they are in ether, dichloromethane, and other common organic solvent. 3. Amino acids have much larger dipole moment than simple amines or simple acids. 4. Amino acid are less acidic than most carboxylic acid and less basic than most amines. In fact, the acidic part of the amino acid molecule is the – NH3 group not a –COOH group. The basic part is the –COO- group, and not a free –NH2 group. R – COOH

Protein

R – NH2

H3N – CHR – COO-

(Wade, 2013).

2

C. The standart of amino acid in Protein The standard amino acids are 20 common -amino acids that are found in nearly all proteins. The standard amino acids differ from each other in the structure of the side chains bonded to their carbon atoms. All the standard amino acids are Lamino acids (Wade, 2013).

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Picture 4. Standart Amino Acid (Wade,2013). D. Isoelectric Point An amino acid bears a positive charge in acidic solution (low pH) and a negative charge in basic solution (high pH). There must be an intermediate pH where the amino acid is evenly balanced between the two forms, as the dipolar zwitterion with a net charge of zero. This pH is called the isoelectric pH or the isoelectric point, abbreviated pI (Wade, 2013).

Picture 5. Isoelectric Point (McMurry, 2008). More specifically, the pI of any amino acid is the average of the two aciddissociation constants that involve the neutral zwitterion. For the 13 amino acids with a neutral side chain, pI is the average of pKa1 and pKa2. For the four amino acids with either a strongly or weakly acidic side chain, pI is the average of the two lowest pKa values. For the three amino acids with a basic side chain, pI is the average of the two highest pKa values.

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4

E. Reactions of Amino Acids 1. Esterification of the Carboxyl Group Like monofunctional carboxylic acids, amino acids are esterified by treatment with a large excess of an alcohol and an acidic catalyst (often gaseous HCl). Under these acidic conditions, the amino group is present in its protonated NH3+ form, so it does not interfere with esterification. The following example illustrates esterification of an amino acid.

Picture 6. Esterification of an amino acid. Esters of amino acids are often used as protected derivatives to prevent the carboxyl group from reacting in some undesired manner. Methyl, ethyl, and benzyl esters are the most common protecting groups. Aqueous acid hydrolyzes the ester and regenerates the free amino acid. Benzyl esters are particularly useful as protecting groups because they can be removed either by acidic hydrolysis or by neutral hydrogenolysis (“breaking apart by addition of hydrogen”). Catalytic hydrogenation cleaves the benzyl ester, converting the benzyl group to toluene and leaving the deprotected amino acid. Although the mechanism of this hydrogenolysis is not well known, it apparently hinges on the ease of formation of benzylic intermediates (Wade, 2013).

2. Reaction with Ninhydrin Ninhydrin is a common reagent for visualizing spots or bands of amino acids that have been separated by chromatography or electrophoresis. When ninhydrin reacts with an amino acid, one of the products is a deep violet, resonance-stabilized anion called Ruhemann’s purple. Ninhydrin produces this same purple dye regardless of the structure of the original amino acid. The side chain of the amino acid is lost as an aldehyde (Wade, 2013).

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Picture 7. Reaction with Nynhydrin (Wade, 2013). F. Peptide Structure The most important reaction of amino acids is the formation of peptide bonds. Amines and acids can condense, with the loss of water, to form amides. Industrial processes often make amides simply by mixing the acid and the amine, then heating the mixture to drive off water (Wade, 2013).

The amide nitrogen is no longer a strong base, and the C-N bond has restricted rotation because of its partial double-bond character. In a peptide, this partial double bond character results in six atoms being held rather rigidly in a plane. Having both an amino group and a carboxyl group, an amino acid is ideally suited to form an amide linkage. Under the proper conditions, the amino group of one molecule condenses with the carboxyl group of another. The product is an amide called a dipeptide because it consists of two amino acids. The amide linkage between the amino acids is called a peptide bond. A peptide is a compound containing two or more amino acids linked by amide bonds between the amino group of each amino acid and the carboxyl group of the neighboring amino acid.

Picture 8. Peptide Bond (Wadde, 2013 ) G. Protein Proteins may be classified according to their chemical composition, their shape, or their function. Protein composition and function are treated in detail in a

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biochemistry course. For now, we briefly survey the types of proteins and their general classifications. Proteins are grouped into simple and conjugated proteins according to their chemical composition. Simple proteins are those that hydrolyze to give only amino acids. All the protein structures we have considered so far are simple proteins. Examples are insulin, ribonuclease, oxytocin, and bradykinin. Conjugated proteins are bonded to a nonprotein prosthetic group such as a sugar, a nucleic acid, a lipid, or some other group (Carey, 2000).

Table 1. Classes of Conjugated Proteins. (Wade, 2013). Proteins are classified as fibrous or globular depending on whether they form long filaments or coil up on themselves. Fibrous proteins are stringy, tough, and usually insoluble in water. They function primarily as structural parts of the organism. Examples of fibrous proteins are in hooves and fingernails, and collagen in tendons. Globular proteins are folded into roughly spherical shapes. They usually function as enzymes, hormones, or transport proteins. Enzymes are protein-containing biological catalysts; an example is ribonuclease, which cleaves RNA. Hormones help to regulate processes in the body. An example is insulin, which regulates glucose levels in the blood and its uptake by cells. Transport proteins bind to specific molecules and transport them in the blood or through the cell membrane. An example is hemoglobin, which transports oxygen in the blood from the lungs to the tissues (Wade, 2013). 1. Levels of Protein Structure 

The primary structure The primary structure of a protein is simply the amino acid sequence (McMurry,). This definition includes the sequence of amino acids, together with any disulfide bridges. All the properties of the protein are determined, directly or indirectly, by the primary structure. Any folding, hydrogen bonding, or catalytic activity depends on the proper primary structure (Wade, 2013).

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

The secondary structure The secondary structure of a protein describes how segments of the peptide (McMurry, ). In particular, the carbonyl oxygen atomsform hydrogen bonds with the amide hydrogens. This tendency leads to orderly patterns of hydrogen bonding: the helix and the pleated sheet. These hydrogen-bonded arrangements, if present, are called the secondary structure of the protein (Wade, 2013).



The tertiary structure The tertiary structure of a protein is its complete three-dimensional conformation. Think of the secondary structure as a spatial pattern in a local region of the molecule. Parts of the protein may have the structure, while other parts may have a-helical the pleated-sheet structure, and still other parts may be random coils. The tertiary structure includes all the secondary structure and all the kinks and folds in between (Wade, 2013).



The Quarternary Structure Quaternary structure refers to the association of two or more peptide chains in the complete protein. Not all proteins have quaternary structure. The ones that do are those that associate together in their active form. For example, hemoglobin, the oxygen carrier in mammalian blood, consists of four peptide chains fitted together to form a globular protein (McMurry, 2008).

2. Denaturation of Protein For a protein to be biologically active, it must have the correct structure at all levels. The sequence of amino acids must be right, with the correct disulfide bridges linking the cysteines on the chains. The secondary and tertiary structures are important, as well. The protein must be folded into its natural conformation, with the appropriate areas of helix and pleated sheet. For an enzyme, the active site must have the right conformation, with the necessary side-chain functional groups in the correct positions. Conjugated proteins must have the right prosthetic groups, and multichain proteins must have the right combination of individual peptides. With the exception of the covalent primary structure, all these levels of structure are maintained by weak solvation and hydrogen-bonding forces. Small changes in the environment can cause a chemical or conformational change resulting in denaturation: disruption of the normal structure and loss of biological activity. Many factors can cause denaturation, but the most common ones are heat and pH (Wade, 2013) a) Reversible and Irreversible Denaturation

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When a protein is subjected to an acidic pH, some of the side-chain carboxyl groups become protonated and lose their ionic charge. Conformational changes result, leading to denaturation. In a basic solution, amino groups become deprotonated, similarly losing their ionic charge, causing conformational changes and denaturation. Milk turns sour because of the bacterial conversion of carbohydrates to lactic acid. When the pH becomes strongly acidic, soluble proteins in milk are denatured. and precipitate. This process is called curdling. Some proteins are more resistant to acidic and basic conditions than others. For example, most digestive enzymes such as amylase and trypsin remain active under acidic conditions in the stomach, even at a pH of about 1. In many cases, denaturation is irreversible. When cooked egg white is cooled, it does not become uncooked. Curdled milk does not uncurdle when it is neutralized (Wade, 2013). Denaturation may be reversible, however, if the protein has undergone only mild denaturing conditions. For example, a protein can be salted out of solution by a high salt concentration, which denatures and precipitates the protein. When the precipitated protein is redissolved in a solution with a lower salt concentration, it usually regains its activity together with its natural conformation (Wade, 2013 ). b) Denaturation cuses heat Heat can be used to disrupt hydrogen bonding and non-polar hydrophobic interactions. This happens because high temperatures can increase the kinetic energy and cause protein molecules to move or vibrate very quickly, thus disrupting the bonds of these molecules. The egg protein is denatured and coagulated during cooking. Some foods are cooked to denature the protein contained in order to facilitate digestive enzymes in digesting the protein (Ophart, C.E., 2003). The heating will make the denatured protein so the ability to bind the water decreases. This happens because heat energy will lead to disconnection of noncovalent interactions that exist in the natural structure of the protein but does not break its covalent bond in the form of peptide bonds. This process usually takes place at a narrow temperature range (Ophart, C.E., 2003). c) Denaturation causes acids and bases The protein will experience the biggest turbidity when it reaches the isoelectric pH, where the protein has the same positive and negative charge, at which time the protein undergoes denaturation marked by increased turbidity and

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incidence of clots. (Anna, P, 1994). Acids and bases can disrupt salt bridges in the presence of ionic charges. A double-replacement reaction type occurs when positive and negative ions in the salt alternate with positive and negative ions from the added acid or base. This reaction occurs in the digestive system, when gastric acid coagulates the milk consumed (Ophart, C.E., 2003). d) Denaturation cause heavy metal Heavy metal salts denature the same protein as acid and base. Heavy metal salts generally contain Hg2 +, Pb2 +, Ag + Ti +, Cd2 + and other metals with large atomic weights. The reaction that occurs between heavy metal salts will result in the formation of insoluble metal-protein salts (Ophart, C.E., 2003). Proteins will experience precipitation when reacting with metal ions. Precipitation by positive ions (metal) is required ph above solution pi because the protein is negatively charged, precipitation by negative ions is required ph solution below pi because the protein is positively charged. Positive ions that can precipitate proteins are; Ag+, Ca2+, Zn2+, Hg2+, Fe2+, Cu2+ and Pb2+, whereas negative ions that precipitate proteins are; salicylate ions, trichloracetate, pichtrates, tannins and sulfosalisilate. (Anna, P., 1994). e) Denaturation cause the addition of chemical compounds Can be caused by chemical reaction between the existing groups with the added compound. For example, the addition of formaldehyde will react to the amino group in the protein with the aminodimethyl acid. The result of this reaction gives a precipitate that is not soluble in water and hardened. (Anwar, et al. 1994). 3. Precipitation of Protein The presence of various functional groups (NH2, NH, OH, CO) and the form of double ions (zwitter ion) contained in the protein structure can lead to the reaction of protein precipitation. The functional groups are capable of binding water molecules through the formation of hydrogen bonds. Deposition reaction may occur due to the addition of chemicals such as salts and organic solvents which can change the solubility of proteins in water (Hidajati, et al. 2017). a) Precipitation with ammonium sulfate The precipitation caused by the addition of concentrated ammonium sulfate causes the dehydration of proteins (water loss). As a result of this dehydratation process, protein molecules that have the smallest solubility will easily settle. Protein deposited in this way does not undergo chemical changes so that it can be

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easily reconstituted by adding water. Deposition in this way is reversible (Hidajati, et al. 2017). b) Precipitation due to concentrated mineral acids Treatment of concentrated mineral acids in proteins can lead to the formation of salt compounds from acidic reactions with amino groups of proteins. Another effect can be irreversible denaturation and protein precipitation is obtained. However, in general, deposition with the addition of strong mineral acids (except concentrated HNO3) is reversible (Hidajati, et al. 2017). c) Precipitation of protein by heavy metals The basis of the precipitation reaction by heavy metals is the neutralization of the charge. Precipitation may occur when the protein is in a negatively charged isoelectric form. Given the positive charge of heavy metals there will be a neutralization reaction of the protein and a salt of precipitated proteinic salt. The precipitate of this protein will dissolve again in the addition of alkali (eg NH3 and NaOH). The precipitation properties of these proteins are reversible (Hidajati, et al. 2017). 4. Color Reaction of Protein a) Biuret Reaction Biuret reactions are common color reactions used for peptide (-CO-NH-) and protein groups. The positive reaction is characterized by the formation of purple color due to the formation of complex compounds between Cu2 + and N of peptide bond molecules. The amount of amino acid attached to the peptide bond affects the color of this reaction. The compound with dipeptide provides blue, purple tripeptide and tetrapeptide as well as a complex peptide giving it a red color. Some proteins that have clusters -CS-NH, -CH-NH- in their molecules also provide a positive color test of this biuretic reaction forming a complex compound illustrated below:

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Picture 9. Complex stucture of Cu2+ peptide compound (Hidajati, et al. 2017). b) Ksanthoprotein reaction Ksanthoprotein color reactions can occur due to the nitration reaction of the benzene ring of the protein amino acids. The test is said to be positively indicated by the yellow color caused by the formation of a polynitrobenzene compound of the amino acid protein. This reaction is positive for proteins containing amino acids with benzene nuclei such as tyrosine, phenyl alamin, tryptophan. In addition the yellow-colored alkali compounds will disappear and turn yellow to orange due to the acidity of the phenol reacting with the alkali. This orange color when acidified will turn back to yellow (Hidajati, et al. 2017). c) Ninhydrin Reaction The color reaction of the ninhydrin protein is positive when it gives color blue or purple. This reaction occurs in the amino-free group of amino acids with the ninhydrin listed below: The above-mentioned blue-violet color can also be used to determine amino acids quantitatively by measuring their absorbance at 570 nm. The basis of this reaction is used in the tool for the determination of amino acids (Hidajati, et al. 2017).

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Picture 10. Ninhydrin Reaction d) Millon's reaction Millon reagents involve the addition of Hg compounds into proteins so that the addition of these metals will produce white deposits of mercury compounds. For proteins containing tyrosine or tryptophan the addition of Millon reagents results in a red color. However, this reagent is not specific because it also gives a positive test of red color in the presence of phenol compounds (Hidajati, et al. 2017). e) Hopkin-Cole reaction The color reaction of these proteins shows positive when marked the formation of purple rings on the boundary plane between protein and reagent solution. The formation of this ring is due to the formation of condensation of 2 indol nuclei from tryptophan with aldehyde. The disinidated aldehyde is obtained by glycosalic acid used for the Adamkiewicz Hopkins test (Hidajati, et al. 2017). 5. Hydrolisis of Protein The addition of alkali in proteins can lead to the hydrolysis of peptide bonds of the protein polymer. This hydrolysis produces amino acid monomers and some amino groups that turn ammonia. Due to the hydrolysis the amount of amino groups decreases. If in proteins there are amino acids that have S atoms such as cysteine and cystine in their molecules then these amino acids can be eliminated into the form of H2S compounds. the addition of Pb salt in an alkaline atmosphere to an easily observed PbS precipitate. Pb2+ + 4OH- → PbO22- + 2 H2O S2- + 2 H2O + PbO22- → PbS ↓ + 4OH-

Protein

(Hidajati, et al. 2017).

13

V. Tools and Material Tool: 

Test tube

20 pieces



Beaker glass 50 mL

2 pieces



Beaker glass 100 mL

2 pieces



Beaker glass 250 mL

1 piece



Bunsen burner

1 piece



Tripod

1 piece

Materials : 

Egg

enough



Milk

enough



Acetate acid

10 mL



(NH4)2SO4 solution

5 mL



NaOH solution

50 mL



HNO3 solution

5 mL



CuSO4 solution

20 mL



PbSO4 solution

20 mL



HgSO4 solution

5 mL



NaNO2 solution

5 mL



Ammonium sulphate solution

5 mL



Nynhidrin solution

10 mL



Formaldehyde solution

5 mL



Mercurysulphate solution

5 mL



Pb acetate

5 mL



HNO3 solution

5 mL



Kongo indicator

enough



PP indicator

enough

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VI. Lanes Work 1. Denaturation of Protein a. Denaturation because of acetate addition 5 mL protein solution Entered into test tube Added 2 drops acetate acid 1N Shaken it. Flake will be formed Heated in steam bath for 5 minutes Observed the precipitate changes

-

Result b. Denaturation because of heating 2-3 mL of protein solution -

Entered into test tube Heated until 1 minute (the precipitate formed) Cooled Divided it into two test tube

Test tube 1

Test tube 2

-

Added 1-2 drops of (NH4)2SO4

-

Heated it

Result

-

Heated it

Result

c. Denaturation because of Formaldehyde 1-1,5 ml formaldehyde

2 ml aquadest

-

Entered into test tube Added protein solution drop by drop Observed the precipitate

Result

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15

2. Amfoter Characteristic of Protein a. Test in acid condition 3 mL of aquadest Entered into test tube Added 1 drop HCl 1N Added several drops congo indicator Added 2-3 mL of protein solution Noted the color changing

Result

b. Test in base condition 2-3 ml protein solution

3 mL of NaOH 0,1 M Entered into test tube. Added several drops of PP indicator

-

-

Entered into test tube Added drop by drop NaOH 0,1 M Observed the color formed Result

Result 3. Precipitation of Protein a. Precipitation of protein with ammonium sulphate 3-4 mL of protein solution -

Poured into test tube Added 3-4 mL saturated ammonium sulphate Shaken it slowly (the solution become turbid) Added 2-3 mL aquadest Shaken it again.

Result b. Protein precipitation with mineral acid

1 mL of HNO

3

-

Poured into test tube Tilted the tube Added 1-1,5 mL protein solution drop by drop through tube wall Let it stand until the white ring formed as protein precipitate Shaken it Added HNO3 again. Until formed white precipitate Result

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1-2 mL of concentrated HCl Poured into test tube Tilted the tube Added 1-1,5 mL protein solution Taken up the tube again Let it stand until the white ring formed as protein precipitate Shaken it Added HCl again. Formed clear solution

-

Result

c. Protein precipitation with heavy metal 1-1,5 mL Protein solution Poured into test tube Added CuSO4 drop by drop

-

- Shaken it Blue precipitate -

Added CuSO4 drop by drop, shaken it until precipitate dilute Repeated again with salt from Zn, Fe, and Hg Observed the changing process

1-1,5 mL Protein solution -

Poured into test tube Added PbSO4 drop by drop

- Shaken it White precipitate - Added PbSO4 drop by drop, -

-

shaken it until precipitate dilute Repeated again with salt from Zn, Fe, and Hg Observed the changing process

Result

Result 4. Reaction the Color of Protein a. Biuret reaction 3 ml Protein -

Entered into test tube Added 1 ml 40% NaOH Added drop by drop 0,5% CuSO4, the color become red or purple

Result

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b. Xanthoprotein reaction 3 ml Protein solution Entered into test tube Added 1 ml of concentrated HNO3

-

- Heated until formed yellow solution Cooled it - Added amonia until formed orange solution c. Ninhydrin reaction Result Protein solution 0.5% -

Set its pH to 7 Taken 1 ml of this solution Added 10 drops of nynhydrin 0.2% solution Heated on 100˚C for 10 minutes Observed the changes

Result d. Millon reaction 2 ml of protein solution - Entered into test tube - Added 1 ml mercury reagent (1% HgSO4 diluted in 10% H2SO4) -

Heated it, formed yellow solution Cooled it Added 1 drop of 1 % NaNO2 solution

-

Heated it again. Formed red solution

Result e. Hopkine-Cole Reaction 1 ml of protein - Entered into test tube - Added 1 drop of formaldehyde solution - Added 1 drop of mercurysulphate reagent - Added 1 ml concentrated sulfuric acid through the wall of test tube until it formed two layers. There are purple ring, if it shaken all of the solution become purple Result

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18

5. Protein Hydrolysis and Sulphur Identification 1 ml protein solution -

Entered into test tube Added 1 ml of NaOH 40% Heated for 1 minute Added one drop of Pb-acetic formed black solution (indicate Pb)

Result

Protein

19

VII. Observation Result No.

Experiment Procedure

1.

Denaturation of Protein a. Denaturation because of acetate addition

-

5 mL protein solution Entered into test tube Added 2 drops acetate acid 1N Shaken it. Flake will be formed Heated in steam bath for 5 minutes Observed the precipitate changes

Result of the Experiment Before

After

Assumpsion / Reaction

Conclusion

Egg : turbid

Egg +

Protein solution if reacted

Protein in egg and milk is

solution

CH3COOH :

with acetate acid causing

denaturated causes

turbid + white

denaturation and formed

adding acetic acid, it is

precipitate

precipitate

marked white precipitate.

Milk : white solution

Heated : turbid + white precipitate

Result Milk + CH3COOH : white solution + precipitate

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20

b. Denaturation because of heating 2-3 mL of protein solution -

Entered into test tube Heated until 1 minute (the precipitate formed) Cooled Divided it into two test tube

-

Egg : turbid

Egg heated

Protein solution will causing Protein in egg and milk is

solution

until 1 minute : denaturation in high

denaturated causes

turbid and

temperature. Adding

heating process. It is

Milk : white

white

(NH4)2SO4 as protein

marked white precipitate.

solution

precipitate

dehydrator, so protein lost H2O

Milk heated Test tube Test tube 1 - Added 1-2 drops of 2 (NH4)2SO4 -

-

Heated it

Heated it

Result

Result

until 1 minute : white solution

EGG (heated) Tube I + (NH4)2SO4 : turbid solution

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21

Tube II : turbid solution

MILK : Tube I + (NH4)2SO4 : white solution Tube II : white solution c. Denaturation because of Formaldehyde

2 ml aquadest

1-15 ml formaldehyd -

Entered into test tube Added protein solution drop by drop Observed the

Result

Formaldehyd

Formaldehyde

Adding formaldehyde is

Protein in egg and milk is

e : colorless

+ aquadest :

causing amino group in

denaturated caused

solution

colorless

protein reagent, yield amino

formaldehyde compound.

solution

acid dimethyl (formed

It is marked with

protein precipitate) and

precipitate.

Aquadest : colorless

+egg (10

solution

drops) : turbid

protein will be denaturation

+ white precipitate

+milk (27 drops) white +

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22

white precipitate 2.

Amfoter Characteristic of Protein a. Test in acid condition

Aquadest :

Aquadest +

colorless

HCl : colorless

solution

solution

3 mL of aquadest -

Result

Entered into test tube Added 1 drop HCl 1N Added several drops congo indicator Added 2-3 mL of protein solution Noted the color changing

Protein in egg and milk is (aq) +

amino acid compound that have characteristic

H+(aq) ⇄

amphoter compound HCl :

+ cango

colorless

indicator (5

solution

drops) : purple solution (++)

Cango

(aq)

because it is reacted with acid and base, it is

Acid condition When the acid is added, the

maeked with white precipitate that form

properties of the protein act

indicator :

+ egg : pink

orange

solution with

solution

white precipitate

as a base. This is because proteins have amphoteric properties (Anwar, C et al: 1996).

+ milk : pink solution with white precipitate

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23

b. Test in base condition 3 mL of NaOH 0,1 M -

Entered into test tube. Added several drops of PP indicator

Result

NaOH :

NaOH + PP (1

colorless

drop) : pink

solution

solution

PP : colorless

Egg + NaOH :

solution

colorless

H2O(aq)

solution

Bases condition

(aq) + OH-(aq) ⇄

(aq) +

When added bases, the 2-3 ml protein solution -

Entered into test tube Added drop by drop NaOH 0,1 M Observed the color formed

Result

Milk + NaOH : properties of proteins are white solution acidic. This is because proteins have amphoteric Egg + NaOH + properties (Anwar, C et al: PP : pink 1996). (+++) solution

Milk + NaOH + PP: pink (++) solution

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24

3.

Precipitation of Protein a. Precipitation of protein with ammonium sulphate

3-4 mL of protein - Poured into test tube - Added 3-4 mL saturated ammonium sulphate - Shaken it slowly (the solution become turbid) - Added 2-3 mL aquadest - Shaken it again. Result

Egg :

EGG :

(NH4)2SO4(aq) + nH2O(l)

Protein in egg and milk

colorless

+ammonium

 (NH4)2SO4.nH2(aq)

can precipitating when

soluion

sulphate :

added with ammonium

turbid solution

sulphate. Then when

Milk : white solution

added by aquadest the + aquadest +

protein can diluted that is

shake :

showing it the

Ammonium

colorless, little

experiment is reversible.

sulphate :

flake

colorless MILK : Aquadest :

+ammonium

colorless

sulphate :

solution

white solution

HNO3 :

+ aquadest +

colorless

shake :; on

solution

bottom colorless, on top white solution

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25

b. Protein precipitation with mineral acid 1 mL of HNO3 -

Poured into test tube Tilted the tube Added 1-1,5 mL protein solution drop by drop through tube wall Let it stand until the white ring formed as protein precipitate Shaken it Added HNO3 again. Until formed

HNO3 :

HNO3 + egg :

Test with concentrated

Protein of egg and milk

colorless

formed ring

HNO3

can formed ring (white) when added by HNO3,

HCl : colorless

+ NO3-

+ HNO3 : yellow precipitate,

 NO3- +

This experiment is irreversible because

Test with concentrated HCl

white precipitate

remain of precipitate containing of in tube.

HNO3 + milk :

Result

added HNO3 again, this precipitate can diluted.

bottom colorless

when shaken it and

+ Cl- 

formed ring 1-2 mL of concentrated HCl -

Poured into test tube Tilted the tube Added 1-1,5 mL protein solution Taken up the tube again Let it stand until the white ring formed as protein precipitate Shaken it Added HCl again. Formed clear solution

Result

Protein

+HNO3 :

Cl- +

yellow precipitate and the bottom colorless

HCl + egg : formed white 26

ring

+ HCl : colorless (30 drops)

HCl + milk : formed white ring

+ HCl : colorless (±200 drops)

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27

c. Protein precipitation with heavy metal

CuSO4 : blue

Egg + CuSO4 :

solution

blue

1-1,5 mL Protein solution Poured into test tube Added CuSO4 drop by drop

-

- Shaken it Blue precipitate -

Protein of egg and milk can forming precipitate if

precipitate

+ Cu2+ ⇌

PbSO4 :

heated some heavy metal (Cu, Pb, Zn, Fe, and Hg).

colorless

+ CuSO4 (5

Then if added again with

solution

drops) :

the some heavy metal,

diluted, blue

then it diluted. That is

Added CuSO4 drop by drop, shaken it until precipitate dilute Repeated again with salt from Zn, Fe, and Hg Observed the changing process

With CuSO4

ZnSO4 :

solution

colorless solution

Result

showing if this With PbSO4

Milk + CuSO4 + Pb2+ ⇌

: blue FeSO4 : little

experiment is reversible.

precipitate

orange solution

+ CuSO4 (20 drops) :

HgSO4 :

diluted, blue

colorless

solution

With ZnSO4

+Zn2+ ⇌

soluttion Egg + PbSO4 : colorless precipitate

Protein

With FeSO4 28

+ PbSO4 (3

+Fe2+ ⇌

drops) : colorless solution With HgSO4 Milk + PbSO4 : white precipitate

+Hg2+ ⇌

+ PbSO4 (3 drops) : white solution

Egg + ZnSO4 : white precipitate

+ ZnSO4 (3 drops) : diluted solution

Protein

29

Milk + ZnSO4 : white precipitate

+ZnSO4 (3 drops) : diluted solution

Protein

30

4.

Reaction the Color of Protein a. Biuret reaction

Egg :

Egg solution +

CuSO4 (aq) + 2 NaOH (aq)

Protein in egg solution

colorless,

NaOH solution

→ Cu(OH)2 (aq) + Na2SO4

and milk can formed

little turbid

: colorless

(aq)

purple solution if reacted

solution

Cu(OH)2 (aq) → Cu2+

with biuret reaction

3 ml

-

Entered into test tube Added 1 ml 40% NaOH Added drop by drop 0,5% CuSO4, the color become red or purple

+2OH-

Milk : white solution

Milk + NaOH : white solution Cu2+

NaOH 40% : Result

colorless

+ CuSO4 (8 drops): purple,

CuSO4 0.5%:

turbid

colorless

b. Xanthoprotein reaction 3 ml Protein - Entered into test tube - Added 1 ml of concentrated HNO3 -

Heated until formed yellow solution Cooled it Added amonia until formed orange solution

HNO3

Egg + HNO3 :

Protein in egg and milk

concentrated

light yellowish

contain amino acid wih

: colorless

solution

benzene group that + HNO3

indicate with orange

+ heated :

solution in xanthoprotein

yellow and

reaction

white solution

Result

Protein

31

+ ammonia : orange solution

Milk + HNO3 : light yellowish solution

+ heated : yellow and white solution, turbid

+ ammonia : orange solution, turbid

Protein

32

c. Ninhydrin reaction Protein solution 0.5% -

Set its pH to 7 Taken 1 ml of this solution Added 10 drops of nynhydrin 0.2% solution Heated on 100˚C for 10 minutes Observed the changes

Ninhydrin

Egg +

Protein in egg and milk

0.2 % :

ninhydrin 0.2

contain α-amino acid that

colorless

% : colorless

indicate from the color

solution

blue purpleish with +

Millon reagent :

+ heated : blue

colorless

purpleish (++)



solution

Result

ninhydrin reaction.

Milk + NaNO2 1 % :

ninhydrin 0.2

colorless

% : turbid, white solution

+ heated : blue

+

+ CO2 + H2O

purpleish

Possitive reaction marked by formed Ruhemann purple that indicate free alpha amino acid (Fessenden, 1986).

Protein

33

d. Millon reaction 2 ml of protein solution -

Entered into test tube Added 1 ml mercury reagent (1% HgSO4 diluted in 10% H2SO4)

-

Heated it, formed yellow solution Cooled it Added 1 drop of 1 % NaNO2

-

solution Heated it again. Formed red solution

Result

H2SO4 :

Egg + millon

Protein in egg and milk

colorless

reagent : white

contain amino acid

solution

turbid and

+ Hg2+ 2H+ + 2NO2- 

(tyrocin and triptofon)

colorless HgSO4 :

solution

colorless solution

+ heated : red precipitate

Millon test identyfy if

NaNO2 1 % : colorless

+ 2H2O

+ NaNO2 : red

theprotein contain tyrosin

solution and

that indicated by red

have flake

complex (Poedjiadi, 2007).

Milk + millon reagent : white turbid solution + heated : red precipitate + NaNO2 : red solution and red precipitate

Protein

34

e. Hopkine-Cole Reaction 1 ml of protein solution -

-

Entered into test tube Added 1 drop of formaldehyde solution Added 1 drop of mercurysulphate reagent Added 1 ml concentrated sulfuric acid through the wall of test tube until it formed two layers. There are purple ring, if it shaken all of the solution become purple

Result

Formaldehyd

Egg +

Protein in milk and egg

e : colorless

formaldehyde :

not contain indol groups

solution

white solution,

because the color not

turbid

became purple ring. +

Millon reagent :

+ millon :

colorless

white solution,

solution

turbid

+ H2SO4 concentrated : grey solution



Possitive reaction marked with form ring with the color is purple, its indicate if the protein contain indol

Milk +

groups.

formaldehyde : white solution, turbid

+ millon : white solution, turbid

Protein

35

+ H2SO4 concentrated : grey (++) solution in the bottom tube and white precipitate in the top of tube

5.

Protein Hydrolysis and Sulphur Identification

1 ml protein solution - Entered into test tube - Added 1 ml of NaOH 40% - Heated for 1 minute - Added one drop of Pb-acetic formed black solution (indicate Pb) Result

Egg :

Egg + NaOH

Pb2+(aq) + 4OH-(aq) →

Amino acid in egg

colorless

40%: colorless

PbO22-(aq) + H2O(l)

protein have more atom

little turbid

solution

S, because react with PbS2-(aq) + 2H2O(aq) + PbO22- acetic.

Milk : white

+ heated :

solution

yellowish solution

NaOH 40% :

(aq) → PbS↓(s) + 4OH-(aq) Amino acid in milk Protein that contain amino

protein haven’t more

acid with atom s will give

atom S, because the color

the color is black if react

became green brownish

colorless

+Pb-acetic :

solution

brown blackish with Pb acetic

if react with Pb-acetic.

solution Pb-acetic :

Protein

36

colorless

Milk + NaOH

solution

40%: white solution, turbid

+ heated : yellow, turbid solution

+Pb-acetic : green brownish

Protein

37

VIII. Analysis and Discussion The experiment which have do on Tuesday, April 3rd, 2018 with the title is “Understanding Characteristic and Color Reaction” and have purpose there are : 1) to differentiate proteins solubility’s property with reverrsible and irreverrsible, 2) to differentiate denaturation reaction of proteins that caused by acid, salt, salt of heavy metal, and heating based on observation, 3) to understand cause happend of precipitated by proteins, and 4) to identify existance of proteins through color reaction. This experiment use egg and milk as sources of protein. Egg are a source of food that many people use. These food contain proteins, fats, vitamins, and minerals. Meanwhile, milk is a solution that contains proteins. Lactose, certain minerals and vitamins that emulsify fats and casein. Casein can be precipitated by acidifying milk to pH 4,7, for non precipitated protein at pH 4,7 it is present serum (Anwar, et al, 1996). 1. Denaturation of Protein. For first experiment, test about denaturation of proteins. In this experiment, consist of three method to denaturazed proteins, there are acetate addition, by heat, and formaldehyde. First method is acetate addition. Prepare proteins solution, for egg, before used it crack the eggshell then separated between yolk and white. That use for experiment is white egg, take about 5 ml of white egg. After that, the white egg diluted with aquadest until 25 ml. Diluted have purpose to make protein solution, because if we use white egg in direct that’s mean not solution. Because texture of white egg not solution. For milk, can use in direct, not diluted before. Arabic chicken’s egg From 25 ml protein solution of egg, take 5 ml of the solution and entered into test tube, the color of egg solution is colorless with little flake. added by 2 drops of CH3COOH 1 N. The purpose this added is to give acid condition, with it can causes form of protein salt that undissolve. After added CH3COOH, the tube shaken, and result flake. Then the tube heated in steam bath for 5 minutes. This heat have purpose to test the mixture will be formed white precipitate or diluted and become colorless. After waiting, truthly the mixture forming white precipitate, that is showing if the protein denaturazed. Characteristic of protein which denaturazed can be look from some even. One of them is the changes of this physical structure. Proteins which have denaturation

Protein

38

usually occuring less solubility. Molecule layer of hydrophobic part will occur position change from inside to outside, this conditions is continues. Like this protein solution that we test have denaturazed caused by added of acetate acid. Denaturation cause added by acid because zwitter ions in proteins. Proteins can forming zwitter ions structure. Proteins also have isoelectric point, count of positive charge and negative charge is equilibrium. So, that is condition make proteins can denaturazed with forming precipitate and become turbid solution. The mechanism is added by acetate acid will broke salt bridge in proteins. Milk Take 5 ml of milk and entered into test tube the color of milk is white. The tube added by 2 drops of CH3COOH 1 N. The purpose this added is to give acid condition, with it can causes form of protein salt that undissolve. After added CH3COOH, the tube shaken, and result flake. Then the mixture heated in steam bath for 5 minutes. This heat have purpose to test the mixture will be formed white precipitate or diluted and become colorless. After waiting, truthly the mixture After waiting, truthly the mixture forming white precipitate, that is showing if the protein denaturazed. Characteristic of protein which denaturazed can be look from some even. One of them is the changes of this physical structure. Proteins which have denaturation usually occuring less solubility. Molecule layer of hydrophobic part will occur position change from inside to outside, this conditions is continues. Like this protein solution that we test have denaturazed caused by added of acetate acid. Denaturation cause added by acid because zwitter ions in proteins. Proteins can forming zwitter ions structure. Proteins also have isoelectric point, count of positive charge and negative charge is equilibrium. So, that is condition make proteins can denaturazed with forming precipitate and become turbid solution. The mechanism is added by acetate acid will broke salt bridge in proteins. The second method is denaturation because heating. 

Arabic chicken’s egg Take 2 -3 ml of proteins solution from solution which have made up. Egg solution that colorless entered into test tube. Heated until 1 minutes, after heated, egg become turbid and little white flake, then cooled. Next, the tube divided into two test tube. Then give lable for each tube. For test tube 1 of egg solution, added 1 -2 drops of (NH4)2SO4 then heated. The function of (NH4)2SO4 is to give acid

Protein

39

and base condition in solution. Added of acid or base can broke salt bridge in proteins which one of interaction in proteins, if will be broke then denaturation will occur. While, for test tube 2, just heated, not added anything. After each heated, compare the result. So, the result of this experiment is egg become turbid, and more white precipitate on added of (NH4)2SO4 and heating. Denaturation can caused by heated. Heat can broke hydrogen bond in proteins but not disturb its covalent bond. That is caused by increasing temperature will make molecule kinethic energy increas. Beceause this increas will broke hydrogen bond and make entalpi system changes be high. If proteins denaturazed, that is showing if the entrophy increasing too. If highest entrophy occur, then occur denaturation and disorder. That is make proteins for bonding water is decrease and occur koagulation 

Milk Take 2 -3 ml of milk then entered into test tube,the color of milk is white. Heated until 1 minutes, after heated, milk white solution, cooled. Next, each tube divided into two test tube. Then give lable for each tube. For test tube 1 of milk, added 1 -2 drops of (NH4)2SO4 then heated. The function of (NH4)2SO4 is give acid and base condition in solution. Added of acid or base can broke salt bridge in proteins which one of interaction in proteins, if will be broke then denaturation will occur. While, for test tube 2, just heated, not added anything. After each heated, compare the result. So, the result of this experiment is milk become white solution, because the precipitate dilute (just heating) and white precipitate on added of (NH4)2SO4 and heating. Denaturation can caused by heated. Heat can broke hydrogen bond in proteins but not disturb its covalent bond. That is caused by increasing temperature will make molecule kinethic energy increas. Beceause this increas will broke hydrogen bond and make entalpi system changes be high. If proteins denaturazed, that is showing if the entrophy increasing too. If highest entrophy occur, then occur denaturation and disorder. That is make proteins for bonding water is decrease and occur koagulation

Protein

40

For last method is with formaldehyde. 

Arabic Chicken’s egg 1 – 1,5 ml of formaldehyde mix with 2 ml aquadest. Fromaldehyde which mixed by aquadest become colorless solution. Then entered into test tube, after that the tube added by protein solution drop by drop. Then observed the precipitate which occur. For egg, resulting turbid solution and little flake. That is showing if occur denaturation of proteins. The flake which formed is reaction result of amino groups in proteins and aminodimethyl acid. Amino acid that bonding with formaldehyde will reacted by acid, beacuse formaldehyde bonded by amino group forming aminodimethyl acid derivate which causes formed precipitate.



Milk Prepare 1 – 1,5 ml of formaldehyde mix with 2 ml aquadest. Fromaldehyde which mixed by aquadest become colorless solution. Then entered into test tube, after that the tube added by protein solution drop by drop. Then observed the precipitate which occur. For milk, resulting white solution and little white precipitate. That is showing if occur denaturation of proteins. The flake which formed is reaction result of amino groups in proteins and aminodimethyl acid. Amino acid that bonding with formaldehyde will reacted by acid, beacuse formaldehyde bonded by amino group forming aminodimethyl acid derivate which causes formed precipitate.

2. Amphoter Characteristic of Protein For second experiment is about amphoter characteristic of Proteins. In this experiment, separated become two experiment. That is in acid condition and abse condition. Acid condition Arabic Chicken’s Egg First, take 3 ml of aquadest. Then entered into test tube. After that, added 1 drop of colorless HCl 1 N and added several drops of Congo indicatore which red color. The result of mixture is soft blue solution. Added 2 ml of egg solution and it changes the color become soft pink solution. This color changes is showing if protein have amphoter characteristic. Amino acid contains of carboxylate ions (CO2-) and ammonium ion (NH3+) in the same molecule corner. That is make proteins have amphoter characteristic, can

Protein

41

react with acid or base. Added of HCl have the purpose is give acid condition and when dropped by Congo indicatore, the solution changes become soft pink solution, that is show acid characteristic and amphoter of proteins. In certain condition, amino group and carboxylate will netralizing, so the molecule nothing charge, can be called isoelectric. If pH under isoelectric condition, then the charge will be positive charge. Meanwhile, if pH upper isoelectric condition, then the charge will be negative charge or netral. Milk First, take 3 ml of aquadest. Then entered into test tube. After that, added 1 drop of colorless HCl 1 N and added several drops of Congo indicatore which red color. The result of mixture is soft blue solution. Added 2 ml of milk and it changes the color become soft pink solution (+). This color changes is showing if protein have amphoter characteristic. Amino acid contains of carboxylate ions (CO2-) and ammonium ion (NH3+) in the same molecule corner. That is make proteins have amphoter characteristic, can react with acid or base. Added of HCl have the purpose is give acid condition and when dropped by Congo indicatore, the solution changes become soft pink solution, that is show acid characteristic and amphoter of proteins. In certain condition, amino group and carboxylate will netralizing, so the molecule nothing charge, can be called isoelectric. If pH under isoelectric condition, then the charge will be positive charge. Meanwhile, if pH upper isoelectric condition, then the charge will be negative charge or netral. The reaction is : COOH

COO-

H +

H3N

C R

H

+

H3N +

C

H

R

Base condition First step is take 3 ml of NaOH 0,1M, then entered into test tube. After that added several drops of PP indicatore. After added PP, the solution become pink solution. PP indicatore have pH average about 8,3 – 10,0. If with acid, then not changes and still colorless, but if with base, will be changes become pink solution. So, NaOH is truthly base compund and this mixture used to as compared.

Protein

42

Arabic Chicken’s Egg First take 2 ml of protein solution (egg) then entered into test tube. Next, added drop by drops of NaOH 0,1 M colorless. After that observed, and resulting strong pink solution with little flake on bottom. The function of NaOH is to give base condition and to increas pH isoelectric which can make protein have base characteristic. PP indicator which added will bonded by base group NHI, that is causes the solution become pink solution. Milk First take 2 ml of protein solution (milk) then entered into test tube. Next, added drop by drops of NaOH 0,1 M colorless. After that observed, and resulting soft pink color. The function of NaOH is to give base condition and to increas pH isoelectric which can make protein have base characteristic. PP indicator which added will bonded by base group NHI, that is causes the solution become pink solution. The reaction is : COOH

COO-

OH +

H3N

C R

H

-

H3N +

C

H

R

3. Precipitation of Protein For thirth experiment is about Precipitation of proteins. In this experiment separated by three experiment, there are with ammonium sulphate, with mineral acid, and with heavy metal. With ammonium sulphate Arabic Chicken’s Egg Take 2 ml of egg solution then entered into test tube. Then, added 2 ml of saturated ammonium sulphate. Shaken it with slowly until turbid and little white precipitate. After that added 1 ml of aquadest until become colorless solution and little flake. This experiment is reversible. The function of ammonium sulphate is to precipitating proteins, but when it added by more, will dilute precipitate of proteins. Added by ammonium sulphate causes hidraze protein, so litlle soluble protein will precipitating. The proteins precipitated not occur chemical changes, so easy to dissolve in water.

Protein

43

Milk Take 2 ml of egg solution then entered into test tube. Then, added 2 ml of saturated ammonium sulphate. Shaken it with slowly until white and little precipitate. After that added 1 ml of aquadest until become colorless in bottom and white solution on top. This experiment is reversible. The function of ammonium sulphate is to precipitating proteins, but when it added by more, will dilute precipitate of proteins. Added by ammonium sulphate causes hidraze protein, so litlle soluble protein will precipitating. The proteins precipitated not occur chemical changes, so easy to dissolve in water. The reaction is :

With mineral acid This experiment separated again become two kind of mineral acid, there are HNO3 and HCl concentrate. With HNO3 Arabic Chicken’s Egg Take 1 ml of HNO3 then entered into test tube. Tilted the tube, after that added 1 ml of egg solution drop by drops through tube wall. Let it stand until the white ring formed as protein precipitate. But, if it cooled, the ring will changes become yellow color. Then, shaken it. After that added HNO3 again and make the precipitate more over than before. This experiment is irreverrsible because added by HNO3. HNO3 concentrate causes of formed salt compound by acid reaction with amino group of proteins. Milk Take 1 ml of HNO3 then entered into test tube. Tilted the tube, after that added 1 ml of milk drop by drops through tube wall. Let it stand until the white ring formed as protein precipitate. But, if it cooled, the ring will changes become little yellow color under ring. Then, shaken it. After that added HNO3 again and make the precipitate more over than before. This experiment is irreverrsible because added by HNO3. HNO3 concentrate causes of formed salt compound by acid reaction with amino group of proteins.

Protein

44

The reaction is :

With HCl Arabic Chicken’s Egg Take 1 ml of HCl concentrate then entered into test tube. Tilted the tube, after that added 1 ml of egg solution drop by drops through tube wall. Let it stand until the white ring formed as protein precipitate. Then, shaken it. After that added HCl again and make the egg solution become colorless. Count of drops which needed about 30 drops. This experiment is reverrsible because added by HCl. HCl concentrate causes of proteins denaturazed by result precipitate that easy to dissolve again. Milk Take 1 ml of HCl concentrate then entered into test tube. Tilted the tube, after that added 1 ml of milk drop by drops through tube wall. Let it stand until the white ring formed as protein precipitate. Then, shaken it. After that added HNCl again and make the milk become colorless. Count of drops which needed about 200 drops. This experiment is reverrsible because added by HCl. HCl concentrate causes of proteins denaturazed by result precipitate that easy to dissolve again. The reaction is :

With heavy metal The principle of the reaction is netralizing charges. The precipitation can occur if protein in isoelectric from that have negative charge. With positive charge of heavy metal, will occur netralizing reaction by protein and resulted netral salt protein which precipitating.. protein precipitate will back to dissolve when added by alkalyne (as NH3 and NaOH). This characteristic of protein precipitations is reverrsible.

Protein

45

In this experiment, do in five kind of heavy metal. Consist of CuSO4, PbSO4, ZnSO4, FeSO4 and HgSO4. For CuSO4 Arabic Chicken’s Egg Take 1 ml of egg solution, entered into test tube. Then added CuSO4 drop by drops (need 3 drops when experiment which have do it). The color CuSO4 is light blue solution. Shaken it until formed blue precipitate. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added CuSO4 drop by drop (need 5 drops when experiment which have do it) to dilute the precipitate (the sample) and result blue solution. Milk Take 1 ml of milk, entered into test tube. Then added CuSO4 drop by drops (need 3 drops when experiment which have do it). ). The color CuSO4 is light blue solution. Shaken it until formed little blue precipitate, but the precipitate which resulted just little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added CuSO4 drop by drop (need 20 drops when experiment which have do it) to dilute the precipitate (the sample) and result blue solution. The reaction is : O R2 H N CH C 2 2 N C COOR H (aq) H

+ CuSO4 (aq)

O R2 H N CH C Cu 2 N C COO R H H

+

SO42- (aq)

2 (s)

For PbSO4 Arabic Chicken’s Egg Take 1 ml of egg solution, entered into test tube. Then added PbSO4 drop by drops (need 3 drops when experiment which have do it). The color of PbSO4 is

Protein

46

colorless. Shaken it until formed white precipitate but the precipitate very little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added PbSO4 drop by drop (need 3 drops when experiment which have do it) to dilute the precipitate (the sample) and result colorless solution. Milk Take 1 ml of milk, entered into test tube. Then added PbSO4 drop by drops (need 3 drops when experiment which have do it). The color of PbSO4 is colorless. Shaken it until formed white precipitate, but the precipitate which resulted just little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added PbSO4 drop by drop (need 3 drops when experiment which have do it) to dilute the precipitate (the sample) and the result is white solution. The reaction is : O R2 H N CH C 2 2 N C COOR H (aq) H

+ Pb(OAc)2 (aq)

O R2 Pb H2N CH C N C COO R H H

+

2 OAc- (aq)

2 (s)

For ZnSO4 Arabic Chicken’s Egg Take 1 ml of egg solution, entered into test tube. Then added ZnSO4 drop by drops (need 3 drops when experiment which have do it). The color ZnSO4 is colorless solution. Shaken it until formed white precipitate. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions.

Protein

47

Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added ZnSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result turbid solution. Milk Take 1 ml of milk, entered into test tube. Then added ZnSO4 drop by drops (need 3 drops when experiment which have do it). ). The color ZnSO4 is colorless solution. Shaken it until formed white precipitate, but the precipitate which resulted just little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added ZnSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result colorless solution. The reaction is : O R2 2 H2N CH C N C COOR H (aq) H

+ ZnSO4 (aq)

O R2 Zn H2N CH C N C COO R H H

+

SO42- (aq)

2 (s)

For FeSO4 Arabic Chicken’s Egg Take 1 ml of egg solution, entered into test tube. Then added FeSO4 drop by drops (need 3 drops when experiment which have do it). The color FeSO4 is light orange solution. Shaken it until formed orange precipitate. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added FeSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result orange solution.

Protein

48

Milk Take 1 ml of milk, entered into test tube. Then added FeSO4 drop by drops (need 3 drops when experiment which have do it). ). The color FeSO4 is orange solution. Shaken it until formed white precipitate, but the precipitate which resulted just little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added FeSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result light white-yellow solution. The reaction is : O R2 2 H2N CH C N C COOR H (aq) H

+ FeSO4 (aq)

O R2 Fe H2N CH C N C COO R H H

+

SO42- (aq)

2 (s)

For HgSO4 Arabic Chicken’s Egg Take 1 ml of egg solution, entered into test tube. Then added HgSO4 drop by drops (need 3 drops when experiment which have do it). The color HgSO4 is colorless solution. Shaken it until formed white precipitate. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added HgSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result colorless with little flake. Milk Take 1 ml of milk, entered into test tube. Then added HgSO4 drop by drops (need 3 drops when experiment which have do it). ). The color HgSO4 is colorless solution. Shaken it until formed white precipitate, but the precipitate which resulted just little. The precipitate can formed caused by protein power or amino acid to bonded by metal ions on the upper isoelectric point. This power causes if pH on the

Protein

49

upper of isoelectric point or amino acid will negative charges. That is so can the proteins bonding metal ions which have positive ions. Next step is the precipitate which formed, take the sample of it and move into other test tube. After that, the sample added HgSO4 drop by drop (need 3-5 drops when experiment which have do it) to dilute the precipitate (the sample) and result turbid solution. The reaction is : O R2 2 H2N CH C N C COOR H (aq) H

+ HgSO4 (aq)

O R2 Hg H2N CH C N C COO R H H

+

SO42- (aq)

2 (s)

4. Color Reaction of Protein a. Biuret Reaction Egg In this experiment the first time was to take 3 mL of egg white protein solution that was slightly yellowish and put into a test tube. Then added 1 mL of NaOH 40% colorless solution. Once added NaOH is produced a colorless solution on the test tube. Thereafter, 0.5% CuSO4 solution was added with a solution of blue color dropwise and required 4 drops of CuSO4 to produce a purple solution which showed a positive reaction to the biuret test. The function of the addition of NaOH is to provide an alkaline atmosphere to the protein. While the addition of CuSO4 serves to determine the existence of peptide bonds or not in protein samples. With the formation of purple color in this protein is due to the formation of complex compounds between Cu2 + and N of peptide bond molecules. Where more and more amino acids on the peptide bonds the purple color gets thicker. Milk In this experiment the first time was to take 3 ml of white milk protein solution and put into a test tube. Then added 1 mL of NaOH 40% colorless solution. After added NaOH the resulting solution remains white in the test tube. Thereafter, 0.5% CuSO4 solution was added in the form of blue solution dropwise and required 3 drops of CuSO4 to produce a purple solution which showed a positive reaction to the biuret test. The function of the addition of NaOH is to provide an alkaline atmosphere to the protein. While the addition of CuSO4 serves to determine the existence of peptide bonds or not in protein samples. With the formation of purple color in this protein is due to the formation of complex

Protein

50

compounds between Cu2+ and N of peptide bond molecules. Where more and more amino acids on the peptide bonds the purple color gets thicker. Biuretic reactions are a common color reaction to test for the presence of peptide groups in proteins. The positive reaction is characterized by the formation of purple color due to the formation of complex compounds between Cu2+ and N from peptide bond molecules. In our experiment, the egg white protein solution yielded a deeper purple color than milk protein, so the egg protein contains more peptides than milk proteins. The equation of the reaction is CuSO4 (aq) + 2 NaOH (aq) → Cu(OH)2 (aq) + Na2SO4 (aq) Cu(OH)2 (aq) → Cu2+ +2OH-

Cu2+

b. Xantoprotein reaction Egg In this experiment the first time was to take 3 ml of egg white protein solution and put into the test tube. Then 1 mL concentrated HNO3 concentrated solution was added. After the added concentrated HNO3 produced a white sediment and a little yellow colored solution. Then the test tube is heated to a water bath and produces a yellow solution and there is a spreading white sediment. In this experiment, nitration reacts to the benzene nucleus of amino acids found in protein molecules by nitro groups. The nucleus of benzene can be nitrated by concentrated HNO3 and produce nitrobenzene derivatives. So the addition of concentrated HNO3 was functioned to minify the benzene core of amino acids. The next step is the reaction tube allowed to cool. Then after cool, NH3 added dropwise and produced an orange solution. To produce orange color is required as much as 2 drops of NH3. On the addition of NH3 the yellow color turns to orange. This is due to the acidity of phenol reacting with ammonia. Milk In this experiment the first time was to take 3 ml of white milk protein solution and put into a test tube. Then 1 mL concentrated HNO3 concentrated

Protein

51

solution was added. After the added concentrated HNO3 produced a white sediment and a little yellow colored solution. Then the test tube is heated to a water bath and produces a yellow solution and there is a spreading white sediment. In this experiment a nitration reaction occurs at the benzene nucleus of the amino acid present in the protein molecule by the nitro group. The nucleus of benzene can be nitrated by concentrated HNO3 and produce nitrobenzene derivatives. So the addition of concentrated HNO3 was functioned to minify the benzene core of amino acids. The next step is the reaction tube allowed to cool. Then after cool, NH3 added dropwise and produced an orange solution. To produce the color orange NH3 required as much as 5 drops. On the addition of NH3 the yellow color turns to orange. This is due to the acidity of phenol reacting with ammonia. From this experimental test of xanthoprotein, the positive result is indicated by the yellow color caused by the nitration reaction at the benzene nucleus of the amino acid so that the formation of the polynitrobenzene compound and after the ammonia is added the color becomes orange because the acidity of the phenol reacts with ammonia. On the addition of the yellow alkali compound will disappear and turn into orange due to the acidity of the phenol reacts with the alkali. The equation of the reaction is H2 C

H C NH2

fenilalanin

COOH

+ HNO3

H2 H C C NO2

COOH

NH2

fenilalanin ternitrasi (kuning)

c. Nynhidrin Reaction Egg In this experiment the first time was to take 1 mL of egg white protein solution that was slightly yellowish and put into a test tube. The pH is then adjusted to a precise pH of 7 which is neutral in a way tested with a universal indicator. Add a solution of NaOH if the protein solution is too acidic and add acetic acid solution if it is too alkaline. Thereafter, 10 drops of 0.2% ninhydrin solution were added. After the addition of ninhydrin solution produced a solution of colorless. Then the test tube is heated over a water bath at 100° C for 10 minutes or until a reaction occurs. By heating the resulting purple solution to form a purple ruheman complex. So the protein in the egg contains α-amino acids. Milk

Protein

52

In this experiment first performed is to take 1 mL of milk protein solution that is white and put into a test tube. The pH is then adjusted to a precise pH of 7 which is neutral in a way tested with a universal indicator. Add a solution of NaOH if the protein solution is too acidic and add acetic acid solution if it is too alkaline. Thereafter, 10 drops of 0.2% ninhydrin solution were added. After the addition of ninhydrin solution produced a white solution faded. Then the test tube is heated over a water bath at 100 ° C for 10 minutes or until a reaction occurs. By heating the resulting purple-colored solution forming a purple ruheman complex. So milk protein contains α-amino acid. In the ninhydrin reaction, a positive result is characterized by the formation of a purple or blue solution after heating, indicating that there is an α-amino acid content in the protein. Amino acids react with ninhydrin to form aldehydes with one lower C atom and release NH3 and CO2 molecules Ninhydrin will be reduced and react with NH3. The equation of the reaction is O

H CH

OH

NH2 + OH

COOH

pH = 7 100oC

O Ninhidrin

O

O

N O

+R

COH + CO2 + H2O

O

kompleks ungu Ruheman (biru-ungu)

d. Millon Reaction Egg In this experiment, the first attempt was to take 2 mL of egg white protein solution which was slightly yellowish and put into a test tube. Then 1 mL of colorless millon reagent was added. After added reagents are produced a solution of colorless. Then heated the reaski tube in a water bath and formed a red sediment. then added 1% NaNO2 and reheated. obtained red color with red sediment. This suggests that the proteins in eggs contain tyrosine / tryptophan and also phenol group Milk

Protein

53

In this experiment the first attempt was to take 2 ml of white egg white protein solution and put into a test tube. Then 1 mL of colorless millon reagent was added. After the reagents are added the resulting solution remains white. Then heated the reaski tube in a water bath and formed a red sediment. Then added 1% NaNO2 and reheated. obtained red color with red sediment. This suggests that the proteins in eggs contain tyrosine / tryptophan and also phenol groups. If the protein is reacted with millon will be a positive value marked by the formation of a red complex. At first Hg dissolved in HNO3 oxidized to Hg+. This Hg+ ion further forms a salt with a carboxyl group of tyrosine. When heated the red sediment formed. This happens because the nitric acid that originally serves as a solvent oxidizes Hg+ to Hg2+. At the same time, the amino acid tyrosine ternitrasi. Then the reaction of the formation of HgO red. The formation of the red complex shows the presence of tyrosine / tryptophan and also phenol compounds in milk and egg whites proteins. The equation of the reaction is H HNO3 C COOH

HO

HO

NH2

O N

Hg2+ H C COOH NH2

O O N

O

HO C

H C

H2 C

NH2

O H

NH2

H O

O

Hg2+

C CH C OH H2 O

O N O

kompleks merah e. Hopkine-Cole Reaction Egg In this experiment the first time was to take 1 mL of egg white protein solution that was slightly yellowish and put into a test tube. Then added 1 drops of dilute formaldehyde that is not colored. And a colorless solution was produced on the test tube. Thereafter, 1 drop of mercury sulphate reagent was added to colorless And the resulting solution remains colorless. Then 1 mL of concentrated H2SO4 is added through the tilt reaction tube wall. After the addition of concentrated H2SO4 produced a du layer of white sediment and brown solution where the white deposits that are above the grey solution. This is not in accordance with the theory

Protein

54

that will form two layers, which is visible purple ring and brownish purple solution. Milk In this experiment first performed is to take 1 mL of milk protein solution that is white and put into a test tube. Then added 1 drops of dilute formaldehyde that is not colored. And the resulting solution remains white in the test tube. Thereafter, 1 drop of mercury sulphate reagent was added to yellow. And the resulting solution remains white. Then 1 mL of concentrated H2SO4 is added through the tilt reaction tube wall. After the addition of concentrated H2SO4 produced a du layer of white sediment and a solution of gray. This is not in accordance with the theory that will form two layers, which is visible purple ring and brownish purple solution. The positive result of the reaction in this experiment was the formation of a purple ring on the boundary plane between the protein solution and the reagent. The formation of this ring is due to the formation of condensation of 2 indol cores from tryptophan with aldehyde groups of glyoxylic acid in the atmosphere of sulfuric acid. Tryptophan is the only amino acid that contains the indole group. If the reaction results indicate a purple ring, the protein contains the indole nucleus of tryptophan with aldehyde. The equation of the reaction is O

NH 2

asam amino triptofan

H COOH

H

+ N

H

C

CH2 CHCOOH

O C

NH2

N H

H

H

asam glioksilat

.

5. Hydrolisis of Protein Egg solution In this experiment the first step is to insert 1 mL of a slightly yellowish colored egg protein into the test tube. Then added 1 mL of NaOH 40%. In the test tube produced a turbid solution. The next step is the reaction tube heated above the water bath for 1 minute. Produced an increasingly murky solution. Then added 1 drops of colorless PbSO4. The reaction tube produced a blackish solution (turbid). The function of adding NaOH is to hydrolyze the peptide bonds of the protein polymer and to the hydrolysis to produce the amino acid monomer. In a protein solution it produces a

Protein

55

browm blackish color this is because the S atom reacts acetic acid and forms a PbS precipitate. Milk In this experiment the first step is to insert 1 mL of white milk protein solution into the test tube. Then added 1 mL of NaOH 40%. In the test tube produced a white solution. The next step is the reaction tube heated above the water bath for 1 minute. And the resulting solution is yellow (white top layer). Then, the test tube was added 1 drop of colorless PbSO4. Produce a green brownish. The function of adding NaOH is to hydrolyze the peptide bonds of the protein polymer and to the hydrolysis to produce the amino acid monomer. In a protein solution it produces a blackish color this is because the S atom reacts acetic acid and forms a PbS precipitate. Pb2+(aq) + 4OH-(aq) → PbO22-(aq) + H2O(l) S2-(aq) + 2H2O(aq) + PbO22-(aq) → PbS↓(s) + 4OH-(aq) IX. Discussion In Hopkin-cole reaction experiments there is a mismatch between the results and the theory. Based on the theory, proteins in egg white and milk are formed 2 layers (purple ring). However in our experiment we did not form a purple ring. This is because the reagent H2SO4 used is not in good condition or has been contaminated so as not to be able to get the results that should be. X. Conclusion Based on the experiments we have done, it can be concluded that: 1. Protein in milk and eggs can be denatured by acid addition, this is indicated by the presence of white precipitate in both protein solutions. 2. Proteins may be denatured by heating. Denaturation of milk and egg proteins is indicated by the presence of white precipitate. 3. Protein in milk and eggs denatured due to the addition of formadehid compounds. Denaturation is irreversible characterized by the presence of white precipitate. 4. Proteins can react with acids and can also react with bases. Therefore it can be concluded that the protein is amphoteric. 5. Protein will precipitate by adding solution (NH4)2SO4 and soluble precipitate with addition of reversible aquadest. 6. Precipitation with HNO3 is irreversible while precipitation with HCl is reversible. 7. Precipitation with heavy metals is reversible. Precipitation occurs when the protein is in a negatively charged isoelectric form.

Protein

56

8. In the color test of proteins with biuret reactions, the results show that egg protein and milk contain peptides. As indicated by the change of color to purple (complex) 9. The addition of NaOH will hydrolyze the peptide bonds of the protein polymer and produce amino acid monomers. In a protein sample containing sulfur it is characterized by a blackish-colored solution indicating the presence of PbS precipitate. 10. In the color test of proteins by reaction ksanthoprotein, showed the results that the protein of eggs and milk formed of polynitro benzene compounds of amino acids characterized by the presence of yellow. 11. In the color test of proteins with the reaction of ninhydrin, showed that in egg protein and milk containing amino acids characterized by the formation of blue-violet color 12. In the color test of proteins by millon reaction, showed that in egg protein and milk containing phenol group characterized by red sediment 13. In the color test of proteins with hopkin-cole reactions, it shows that egg protein and milk proteins not contain the indole nuclei of tryptophan with aldehydes characterized by the not formation of purple rings and brownish-purple solutions.

XI. Answer Question 1. Explain what is the function of testing the protein with each test reagent (CuSO4, HgCl2, HNO3, Pb-acetate)! Answer: The function of protein test with each reagent as follows: 

CuSO4 is used to test for the presence of heavy metals in proteins characterized by presence of precipitation when positive proteins contain heavy metals.



HgCl2 is used for the test of a protein containing a phenyl hydroxyl group (-OH).



HNO3 is used to test for the presence of benzene rings from the amino acid salt of the proteins, ie in this experiment when concentrated nitric acid is added and produces nitrobenzene derivatives.



Acetate pb is used to test for the presence of cysteine and methionine amino acids, which in this experiment will produce a black solution because the S atom reacts with acetic acid to form PbS precipitate.

2. How does the effect of organic solvents (acetone and ethanol) on the nature of protein denaturation? Answer:

Protein

57

The effect of organic solvents (acetone, ethanol) on the nature of protein denaturation is that proteins or nucleic acids will lose their secondary and tertiary structures because organic solvents result in denatured proteins. 3. List the various bonds that cause the polypeptide to be stable in alpha-helical form! Answer: 

Disulfide bond Formed between 2 cysteine residues interconnected 2 parts of the polypeptide chain through cysteine residues.



. Hydrogen bond Formed between the NH- or -OH groups and the C = O groups in the peptide or -COO-bond in the R group.

XII. References Anna, P. 1994. Dasar-Dasar Biokimia. Jakarta : Universitas Indonesia. Anwar, Chairil. 1996. Pengantar Praktikum Kimia Organik. Jakarta : DIKTI. Carey, Francis. 2000. Organic Chemistry. United State:McGrawHill. Fessenden, Ralp J dan Joan S Fessenden. 1986. Kimia Organik jilid 2. Jakarta : Erlangga. Hidajati, Nurul, dkk. 2017. Penuntun Praktikum Kimia Organik II. Surabaya: Jurusan Kimia FMIPA Unesa. McMurry, John. 2008. Organic Chemstry. United State of America : Thomson Learning Inc. Ophart, C.E., 2003. Virtual Chembook. Elmhurst College. Poedjiaji A and Supriyanti. 2007. Dasar-Dasar Biokimia. Jakarta: UI Press

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Wade, L G. 2013. Organic Chemistry. United State of America: Pearson Education.

XIII. ATTACHMENT 1. Denaturation of Proteins

Prepare the tools for all experiment

Prepare the materials for all experiment

Formed white precipitate after added 2 drops of CH3COOH 1N

After heated, egg solution become turbid and little flake, milk solution is white

Protein

Protein solution that used is egg and milk, each solution which needed is 5 ml

Prepare 2 -3 ml proteins solution (egg and milk)

59

After heated 1 minutes, egg become turbid and little white flake, while milk white solution

Devided into 2 test tubes, and each one of them heated + (NH4)2SO4, but each other just heated

Formaldehyde + aquadest become colorless solution

Formaldehyde + aquadest + Egg resulting turbid solution and little flake, while and milk resulting white solution and little white precipitate 2. Amphoter characteristic of Protein

HCl + aquadest resulting colorless solution

Protein

Prepare the Congo indicatore

Added Congo indocatore resulting soft blue solution 60

If added by egg result soft pink solution with white precipitate, by milk result pink solution with white precipitate

NaOH + PP indicatore result pink solution

When egg added by NaOH result colorless solution, while milk and NaOH result white solution

When added by (NH4)2SO4, for

After added aquadest and shaken,

When added by PP indicatore, in egg tube become strong pink solution with little flake, in milk tube become soft pink color 3. Precipitation of Protein

Prepare the protein solution, use 2

Protein

61

ml of egg and milk

egg result turbid solution and little white precipitate. Foor milk result white solution and little precipitate

for egg result colorless solution and little flake. For milk result colorless in bottom and white solution on top

HNO3 + egg formed white ring as precipitate, but for a few second become yellow ring. HNO3 + milk formed white ring and little yellow color

When added HNO3 again into each tube, egg or milk still have precipitate while little dissolve

HCl + egg formed white ring, HCl + milk formed white ring too. The ring showing as precipitate

When added HCl again into each tube, egg will be colorless solution (30 drops) and milk will be colorless solution too (200 drops)

Prepare protein (egg) solution 1 ml into 5 test tube

Also prepare the milk entered into 5 test tube each 1 ml

Protein

62

Egg added by some heavy metal (CuSO4, PbSO4, ZnSO4, FeSO4 and HgSO4) After add, all result precipitate, but for PbSO4 just little formed precipitate

Milk added by some heavy metal (CuSO4, PbSO4, ZnSO4, FeSO4 and HgSO4) After add, all result precipitate, but for PbSO4 just little formed precipitate

Take sample from each test tube of egg, then diluted with similar heavy metal which added before CuSO4 (5 drops), PbSO4 (3 drops), ZnSO4 (3-5 drops), FeSO4 (3-5 drops), HgSO4 (3-5 drops)

Take sample from each test tube of milk, then diluted with similar heavy metal which added before CuSO4 (20 drops), PbSO4 (3 drops), ZnSO4 (3-5 drops), FeSO4 (3-5 drops), HgSO4 (3-5 drops) 4. Reaction the Color of Protein

Protein

63

Prepare protein solution, egg and milk

After added NaOH + CuSO4, for egg solution result purple color (++), for milk result purple, turbid

Prepare protein solution, egg and milk

Egg solution + HNO3 result light yellowish solution, milk + HNO3 result light yellowish solution

After heated, egg result yellow and white solution, for milk result yellow and white solution, turbid

When added by ammonia concentrate, for egg result orange solution, for milk result orang solution, turbid

Egg + Nynhidrin + heated result blue purplekish (++), while milk + nynhidrin + heated result blu purplekish

Prepare protein solution, egg and milk

When added millon reagent, for egg result turbid and colorless solution, for milk result white turbid solution

Protein

64

After heated, both of them result red precipitate

When added by NaNO2, for egg result red solution and red precipitate, for milk result red solution and little red precipitate which float up

Prepare protein solution, egg and milk

Egg + formaldehyde + Millon When egg added by H2SO4 reagent result white solution, concentrate result grey solution. turbid. Wile milk + formaldehyde Milk added by H2SO4 concentrate + Millon reagent result white result grey solution in the bottom solution turbid and white precipitate on the top 5. Protein Hydrolisis and Sulphur Identification

Egg + NaOH result colorles, milk + NaOH result white solution turbid

Protein

After heated, egg become yellowish solution, while milk become yellow, turbid solution

Egg + Pb acetic result brown blackish solution. Milk + Pb acetic result reen brownish

65