Salivary Amylase

Activity of Salivary Amylase Domingo, Guray, Hugo, Lorenzo, Mohammad Isa

Intro Because everything has a start

Catalysis

 The process of increasing the rate of reaction with the use of a catalyst.  Catalyst – any substance that increases rate of reaction upon addition to a certain reaction

Page  3

Enzymes

 Act on substrates in a reaction  Highly specific  Breaks down complex macromolecules, synthesizes compounds essential for the cell  Active site  Enzyme-substrate complex  Speeds up reaction rates

Page  4

http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg Page  5

Enzymes

 Require cofactors for activity  Classified according to the types of reaction they catalyze

– Oxidoreductase – Transferase – Hydrolase – Lyase – Isomerase – Ligase

Page  6

Page  7

Amylase

 An enzyme that breaks down starch into oligosaccharides through hydrolysis  Secreted by the human’s parotid glands and the pancreas  α-Amylase  β-Amylase  γ-Amylase

Page  8

Factors that may affect catalysis rates

 Temperature  pH  Enzyme concentration  Amount of substrate

Page  9

Materials and Methods

Solution Preparation

Saliva was collected. 1 ml of saliva was diluted to 10 ml with distilled water.  10 % salivary amylase solution

Page  11

Estimation of salivary amylase activity

A mixture of 5.0 mL 1% starch, 2 mL 1% NaCl solution and 2 mL phosphate buffer put in a test tube and then placed in a water bath At 38oC, 1 mL salivary enzyme solution added to the solution. A drop from the digestion mixture mixed with 1 drop of iodine for every minute.  Achromic point was determined.

Page  12

Effect of enzyme concentration

The salivary amylase solution diluted to five lower concentrations: 2.5%, 2.0%, 1.5%, 1.0%, 0.75% and 0.5%.  The same procedure done as previous using 1% concentration of starch solution. Reaction rates observed for each dilution.

Page  13

Effect of amount of substrate

 Six percent starch prepared from which five other dilutions were prepared: 5%, 4%, 3%, 2%, and 1%. The same procedure for Estimation of salivary amylase activity used using 2.5% salivary amylase solution. Reaction rates for each substrate dilution recorded.

Page  14

Effect of pH

A mixture of 0.2 M sodium biphosphate (Na2HPO4) and 0.1 M citric acid prepared to obtain different buffer solutions with pH varying from 3.0 to 8.0. Similarly, procedures from the estimation of enzymatic activity were applied, recording all notable reaction rates for each pH setup.

Page  15

Effect of temperature

Test tube with 2.5% salivary enzyme was placed on water baths maintained at 4oC, 10oC, 38oC, 58oC, 78oC and 100oC.  Reaction rates recorded.

Page  16

Results and Discussion

Estimation of amylase activity

 Achromic point is the time it takes for the enzyme to completely hydrolyze the starch solution.  enzyme-starch mixture is not able to produce a blue to violet color with iodine -> absence of starch

Page  18

Estimation of amylase activity Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva and Their Corresponding Time to Reach the Achromic Points, Amylase Units and Enzyme Activity Effect of Enzyme Concentration Salivary Amylase (%)

time to achromic point (min)

0.5

30

0.75

23

1

18

1.5

11

2

7

2.5

7

amylase units

Enzyme activity

0.833333333

0.4000

1.630434783

0.2667

2.777777778

0.2000

6.818181818

0.1333

14.28571429

0.1000

17.85714286

0.0800

Amylase units - amount of enzyme necessary to digest 5 ml 1% starch to reach the achromic point within 10 minutes Enzyme activity - mg starch hydrolyzed per minute per unit enzyme Page  19

Effect of Enzyme Concentration

enzyme + substrate <=> enzyme-substrate complex <=> enzyme + product

Page  20

Effect of Enzyme Concentration Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva and Their Corresponding Time to Reach the Achromic Points, Amylase Units and Enzyme Activity Effect of Enzyme Concentration Salivary Amylase (%)

time to achromic point (min)

0.5

30

0.75

23

1

18

1.5

11

2

7

2.5

7

amylase units

Enzyme activity

0.833333333

0.4000

1.630434783

0.2667

2.777777778

0.2000

6.818181818

0.1333

14.28571429

0.1000

17.85714286

0.0800

Figure1. Enzyme Activity with Varying % Concentrations of Salivary Amylase Page  21

Effect of Enzyme Concentration

Increased enzyme concentration increases reaction rate (more enzymes are present to act upon a fixed amount of substrate)

However, as substrate concentration is constant, it produces a limiting effect on reaction rate (excess enzymes begin to compete for substrate)

Surplus of enzymes on a limited reaction rate causes overall enzyme activity to diminish (reaction rate cannot cope up with increased enzyme conc)

Page  22

Effect of Enzyme Concentration

Page  23

Effect of the Amount of Substrate

 Rate of Reaction – describes how fast a chemical reaction proceeds – depends on reactant and product concentrations • More importantly on rate constant k

Page  24

Effect of the Amount of Substrate

 Enzyme Kinetics – still follows the same trend • Increasing either substrate or enzyme increases rate – but there is a limit to this relation • When enzyme conc are constant, there is a limit to the velocity of the reaction

Page  25

Effect of the Amount of Substrate

 Michaelis-Menten Kinetics – enzymatic reactions are observed to reach a maximum rate of reaction Vmax – constant enzyme concentration provides a limiting effect • All enzymes are bound to substrate – vo= Vmax [S] / (Km + [S])

Page  26

Effect of the Amount of Substrate

 Rectangular Hyperbola  Max rate of Vmax  Half-Velocity is reached at Km  Vmax is dependent on [E}  Km is constant – Km=K-1 + K2 / K+1. – Measure of affinity Page  27

Rate of Reaction vs. Substrate Concentration Rate increases with substrate concentration However exponential relation Due to experiment limit (30 min) Km and Vmax not evident Page  28

Rate of Reaction vs. Substrate Concentration Allowing time to go beyond 30, rectangular hyperbola is attained Change in rate diminishes as substrate conc increases Vmax still indiscernible together with Km Page  29

Effect of the Amount of Substrate

 Asymptotical nature makes it hard to determine which much certainty the values of Km and Vmax  Algebraic manipulation (double-reciprocal plot) allows linear expression of MM eq.  Lineweaver-Burke Equation – : 1/vo = (Km / Vmax) (1/ [S]) + (1/Vmax)

Page  30

Effect of the Amount of Substrate

 Linear equation  Regression Analysis allows determination of Km and Vmax  Also able to determine nature of protein function inhibition (competitive, uncompetitive, noncompetitive) Page  31

Double Reciprocal Plot Not strong liinear relation (due to experimental limits) R2 value of only 0.785 Vmax= 4.24719E-05 Page  32

Km= 0.000484646

Double Reciprocal Plot Perfect linear relation Vmax= 2.08333E-05 Km=4.16667E-05 Very Low Km, high affinity of enzyme Vmax close to velocity values, near saturation Page  33

Effect of pH

• Enzymes are proteins – function is ultimately determined by their structure

• optimal pH range – Changes in pH • excess of either H+ or OH- ions • affect the secondary, tertiary and quarternary structures by disrupting hydrogen bonds and van der wal interactions. • change the active site of the enzymes – preventing the enzymatic reaction

Page  34

Effect of pH

• Enzymes – have a range of pH at which it is active and outside of which it is inert – optimum pH • most favorable pH value • point where the enzyme is most active • extremely high or low pH values generally result in complete loss of activity for most enzymes

• Salivary amylase has an optimum pH of around 7

Page  35

Effect of pH

Figure5. Effect of Varying pH in Enzymatic Activity of Amylase. Page  36

Effect of pH

 enzymatic activity of salivary amylase is highest at pH 7 – pH of oral cavity is close to 7

 At pH 8, – decrease in activity

 acidic pH 3, 4 and 5 – enzyme acitivity was at minimum

Page  37

Effect of Temperature

• reaction rate of an enzymatic reaction increases as the temperature is raised • 10° C rise in temperature will increase the activity of most enzymes by 50 to 100% • many enzymes are adversely affected by high temperatures – Reaction rates may increase with temperature up to a maximum level, but then abruptly decline with further increase of temperature

• increases in temperature are able to break H-bonds and van der wal interactions

Page  38

Effect of Temperature

 decrease in temperature, rate of reaction is decreased due to lowered energy  over a period of time, enzymes will be deactivated at even moderate temperatures  most enzymes lose activity at 5°C and when frozen

Page  39

Effect of Temperature

Figure6. Effect of Varying temperature in Enzymatic activity.

Page  40

Effect of Temperature

• highest enzyme activity was seen in the temperature 38°C – near temperature in the oral cavity

• other temperature levels – enzyme activity was found to be minimal – achromic point was not reached within the 30min limit

• optimum enzyme activity is at that level closest to the natural physiological setting • high heat – denaturation

• very low temperatures – lowered chemical kinetics

Page  41

Conclusion

 1 ml of human saliva has around 17.86 Units of amylase.  Increased enzyme concentration increases reaction rate, however, it decreases overall enzyme activity.  Increased substrate concentration increases reaction rate, however there is a maximum rate that can be achieved. (Vmax)  Michaelis-Menten and Lineweaver-Burke describes enzyme kinetics. The Michaelis constant Km gives an idea on enzyme affinity.  Salivary amylase has a Km = 4.84e-4 (corrected: 4.167e-5)  There is an optimal pH and temperature range for enzyme activity. Outside this range, enzyme activity drastically decreases due to denaturation and deactivation.  Optimal pH would be near 7 while optimal temperature should be near 37 C. Page  42

Thank you!! 

Page  43