Midterms Reviewer.docx

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CHEM 40.1 - MIDTERMS REVIEWER

the action of a phosphate buffer against added base or acid is show as:

Discussion: Buffers 

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buffer – a solution that resists drastic changes in pH when small amounts of acid or base are added o regulates the pH of fluids and tissues of living organisms within the limits consistent with life and normal functions most if not all biological processes are tightly regulated by the pH of the medium in which they occur o the conformation and activity of biomolecules as well as the concentrations of molecular and charged species in solution are very sensitive to pH changes metabolic processes continuously alter intracellular H + concentration, and since optimum activities of many biomolecules occur within a limited pH range, living organisms are equipped with remarkably efficient mechanisms to maintain intracellular H+ concentration in humans, these mechanisms include o the buffer systems of the body o the action of the kidneys by which acids and bases are excreted in the urine o the respiratory mechanism by which the H+ concentration in bodily fluids is regulated by the rate of carbon dioxide elimination in the lungs buffers are also used in the laboratory to control the pH of culture media for microorganisms and tissues Buffer Type 1. solutions of weak acid and its salt (or conjugate base) 2. solutions of a primary salt and a secondary salt 3. solutions of a weak base and its salt (or conjugate acid) 4. solutions of ampholytes

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OH-(aq)

+

H2PO4-(aq) Acid

 HPO42-(aq) Conjugate Base

+

H2O

OH-(aq)

+

HPO42-(aq) Base

 H2PO4-(aq) Conjugate Acid

+

H2O

when 2 or more buffers are present, the effects are additive so that the buffering ability occurs over wider range the buffering action of a weak base and its salt is due to the ability of the base component to neutralize the added base o the buffer solutions of weak bases and their salts are not commonly used because of the volatility and instability of the bases and the dependence of their pH on pKw which is markedly affected by temperature changes pseudo buffers – in contrast to the buffering action of weak acids, that of dilute solutions of a strong acid or strong base is due to the amphoteric properties of the solvent, such as water, which can act as either an acid or base some organic buffers with good buffering capacity in the physiologically important pH range of 6 – 8 are used in the biochemistry laboratory o zwitterionic amino acids, mainly N-substituted taurines, or N-substituted glycines

Example HOAc – NaOAc

NaH2PO4 – Na2HPO4 N-(2-acetamido)-iminodiacetic acid (ADA) NH3 – NH4Cl

amino acids and proteins

Buffer Capacity  buffer action – is the resistance of a buffer solution to a change in [H+]  the buffering action of a solution of a weak acid and its salt is due to the salt component to neutralize any added acid, and the ability of the acid component to neutralize any added base  consider the HOAc-NaOAc buffer solution:  OAc-(aq) + Conjugate Base

OH-(aq)

+

HOAc(aq) Acid

H+(aq)

+

OAc-(aq)  Conjugate Base

CHEM 40.1 – Midterms Reviewer

HOAc(aq) Acid

+

N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES)

2-(N-morpholino)-ethanesulfonic acid (MES) H2O

H2O

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Tris-(hydroxymethyl)aminomethane (Tris or THAM) /steffigatdula/

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 N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) Henderson-Hasselbalch Equation  the pH of a buffer solution made up of known concentration of a weak acid and its salt can be calculated using the Henderson-Hasselbalch equation: pH = pK a + log

addition of SA: pH = pK a + log addition of SB: pH = pK a + log

[base] [acid]

mmol base−mmol H+ add. mmol acid+mmol H+ add. mmol base+mmol OH− add. mmol acid−mmol OH− add.

pOH = pK b + log addition of SB: pOH = pK b + log addition of SA: pOH = pK b + log 

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[acid] [base]

mmol acid −mmol OH− add. mmol base+mmol OH− add.

from the Henderson-Hasselbalch equation, calculate the ratio of salt to weak acid required to obtain the desired pH if both acid and salt form as concentrate and/or stock solutions or as solid, mix the 2 forms in calculated amounts ad dilute to a volume just less than desire final volume of the buffer o verify the pH using a pH meter o if necessary, adjust the pH by adding a small amount of 1 M solution of acid or base until the desired pH is attained, then add distilled water to the desired final volume if only the base (or acid) form is available, you will have to generate the acid form first o calculate the amount of base (or acid) corresponding to the total number of moles of buffer needed  mole base (or acid) = moles buffer  mole base (or acid) = (desired conc. of buffer) x (desired volume of buffer) o this is the amount of base (or acid) you will measure out o slowly add small amount of strong acid (or base) to the measured amount of base (or acid) until the pH desired is attained o adjust to the required volume by dilution with distilled water

mmol acid+mmol H+ add.

Activity: Micropipetting – Transferring Minute Volumes

mmol base−mmol H+ add.

ionization of the generalized weak acid HA: HA(aq) Acid

 H+(aq) + Conjugate Base

A-(aq)

[A− ] [HA] the pH of a buffer solution is also influenced by the following factors: o addition of neutral salts – a change in ionic strength changes the pH o dilution – a decrease in ionic strength decreases the reserve acidity or alkalinity of the buffer o temperature – the pKa of a buffer is dependent on the temperature; the pH of a buffer can change as a function of temperature to be suitable for a particular application, the buffer must meet the following requirements: o buffer pH must be within 1 unit of the pKa value of the acid or the base o buffer components must be appreciable soluble in water o buffer components should not react with each other pH = pK a + log

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x= 

General Steps in the Preparation of Buffers  choose an appropriate buffer system o the weak acid component should have a pKa closest to the desired buffer pH to ensure maximum buffer capacity CHEM 40.1 – Midterms Reviewer

mean - average of the values measured from the sample; use a calculator to get this (x) (x1 + x2 + x3 + . . . xn ) n

standard deviation, s – square root of variance; use a calculator to get this (xσn-1); same unit 2

s= √

2

∑ni=1 (xi − x)2 n−1 /steffigatdula/

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RSD = 

Albumin  most abundant protein in egg whites  functions include transport of fatty acids, keeping fluid from leaking out of the circulatory system, pH maintenance

relative standard deviation, RSD s x 100 x

range R = xhighest − xlowest

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20 mL of egg whites + 2.0 mL of 1.0M HOAc, dropwise  filter mixture through damp cheesecloth; press against the sides of the funnel  slowly add an equal volume of saturated (NH4)2SO4 (ammonium sulfate) to the filtrate

Grubb’s Test o used to detect outliers; can only detect 1 outlier per data set o arrange data set from lowest to highest then calculate |xi − x| for both extremes, calculate gexp for the value with a higher |xi − x| o if gtab > gexp then the value is accepted, otherwise it is rejected o if gexp is rejected, calculate the new s and x for the data set with the outlier removed o uses n in the table of critical values g=

max |xi − x|

i=1..n

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s

Experiment 1: Extraction and Isolation of Proteins incubate at room temperature for about 30 minutes  centrifuge the mixture and discard the precipitate  transfer the supernatant into an Erlenmeyer flask immersed in an ice bath  add an equal volume of saturated (NH4)2SO4 until turbidity persists

Extraction  Isolation  Int. Purification  Fin. Purification

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extraction o starts with cell lysis o mechanical and non-mechanical cell disruption  sonication, French press, mortar and pestle o additional reagents can be added to aid extraction  suspension in hypotonic buffer (causes cells to burst)  detergents (may denature proteins)  addition of Glass Beads  addition of Nucleases  lysozyme (dissolves cell walls) o addition of protease inhibitors ensure that the desired proteins will not be hydrolyzed isolation o differential solubility separation techniques  salting in/out, isoelectric precipitation, precipitation by acetone or alcohols intermediate purification o removes most of the bulk impurities (i.e. other proteins and nucleic acids) o IEC, GFC, affinity chromatography, and electrophoretic methods final purification o a highly pure protein is achieved by removing trace impurities and other closely related species o HPLC, isoelectric focusing, 2D-SDS-PAGE

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precipitates unwanted (coagulum)

other components

cell lysis; membrane disruption; homogenization

“salting in”; at low salt concentrations, the solubility of the protein increases slightly; ions from the salt associate with the surface of the protein, this shields those areas from the solvent molecules (water)

separation based on density (heavier particles at the bottom)

“salting out”; at higher salt concentrations, the solubility of the proteins decrease; all the binding sites on the protein surface for the salt ions have become occupied and so the ions begin to interact with the solvent; concentration of "free" solvent molecules decreases as they are used to solvate the salt ions; protein molecules therefore move closer together and begin to interact with one another via the hydrophobic or charged patches on their surfaces; at some salt concentration , the protein molecules aggregate and come out of solution.

allow mixture to stand for 15 minutes  CHEM 40.1 – Midterms Reviewer

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o

centrifuge the mixture; collect precipitate  dissolve with 10 mL 0.9% NaCl; calculate for % w/v

o

Casein  contains all the essential amino acids  precipitated thru isoelectric precipitation (pI = 4.7) o CaCaseinate(aq) + 2H+(aq) → Casein(s) + Ca2+(aq) 15 mL of milk + 0.1M HCl until flocculatent precipitate forms  centrifuge mixture; discard supernatant  add 1 mL 95% ethanol and invert tube to wash residue; decant alcohol; repeat twice  wash precipitate with 1 mL acetone; decant washings and air dry under hood  dissolve precipitate in 10 mL 0.01M NaOH; calculate for % w/v

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 removes adsorbed water

removes adsorbed lipids

Quantitation

Through

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Disadvantages only qualitative/semiquantitative will account only for the free aromatic amino acids in the protein

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Bradford Assay o the binding of Coomassie Brilliant Blue G-250 to protein under acidic conditions causes a shift in the dye’s wavelength of maximum absorption from 465 nm (brown) to 595 nm (blue)

CHEM 40.1 – Midterms Reviewer

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Disadvantages formation of protein-dye complex can be affected by the number of available/free amino acids within the protein, so choosing the protein standard is important

Biuret Assay o colorimetric determination of protein concentration, measures the number of peptide bonds o biuret reagent is made in basic solution using copper sulfate (CuSO4) and potassium tartrate (K2C4H4O6) o the atoms involved in the peptide bond form a complex with Cu2+, forming a violet solution that can be measured at 550 nm Advantages not significantly affected by differences in amino acid composition between proteins, easily detects the presence of peptide bonds

Warburg-Christian Assay o estimates protein concentration based on the absorbance of exposed tyrosine and tryptophan residue o absorbance measured at 280 nm at 260 nm; 260 nm adjusts for the absorbance of nucleic acids (whose absorption spectra overlap with those of tyrosine and tryptophan) o protein conc. (mg/mL) = 1.5(A280)-0.76(A260) o ratio of A280/A260 can estimate the purity; A280/A260 = ~1.75, the higher the % purity Advantages simple, non-destructive, interfering substances can be accounted moderate sensitivity (501000 μg)

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Advantages simple and sensitive, few interferences high sensitivity (~1 μg)

isoelectric focusing; casein precipitates out

Experiment 2: Protein Spectrophotometric Methods

absorption at 545 nm is directly related to the concentration of the protein requires a protein standard and creation of calibration curve  the protein standard should match the structure and reactivity of the extracted protein

Disadvantages lack sensitivity, unsuitable for solutions with protein content of less than 1 mg/mL

Lowry Method o Biuret Assay + Tyr and Trp oxidization by Folin and Ciocalteau reagent producing a bluepurple complex o extremely sensitive (~1 μg/mL) but is subject to interferences from a wide range of non-protein substances o requires external calibration o incubation increases sensitivity Bicinchoninic Assay (BCA) o purple colored product (λmax = 562 nm) o more sensitive than Biuret and Lowry, less variability than Bradford o incubation increases sensitivity o high sensitivity; 1 μg/mL Ninhydrin Method o determines the amount of free amino nitrogen o yellow  deep purple product Kjeldahl Method o used to estimate protein content in foods and other samples o quantitative determination of total N = sum of organic N, ammonia and ammonium

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Experiment 3: Monitoring Protein Conformational Changes by Viscosity and CD Spectroscopy

Experiment 7: Enzyme Kinetics

Denaturants Denaturant

Factor

Molecular Level

HCl

pH

NaOH

pH

Destroys H bonds, affects electrostatic interactions

Heat

Temperature

Destroys weak interaxns, can irreversibly denature protein

NaCl

Ionic Strength

Affects ion bridges; leads to solvation

Urea

Chaotropic Agent

SDS

Detergents

Denatures protein by allowing water to solvate hydrophobic groups Amphiphatic, tail disrupts hydrophobic interaxns

β-MeOH

Reducing Agent

Converts disulfide bridges to sulfuhydril groups

Viscosity  observes alterations in the tertiary and quaternary structures of proteins  viscosity – measure of a liquid’s resistance to flow o denaturation causes protein (albumin) to go from globular to rod-shaped/extended form; viscosity goes up o denaturation of globular proteins causes unfolding of compact tertiary structures into flexible stretched-out amino acid chains  CD-ORD Experiment 4: Ion Exchange Chromatography

Experiment 5: Protein Characterization by Gel Filtration Chromatography Experiment 6: Protein Characterization by Electrophoresis CHEM 40.1 – Midterms Reviewer

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