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

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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

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http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg
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Enzymes

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

Oxidoreductase Transferase Hydrolase Lyase Isomerase Ligase

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Amylase

 An enzyme that breaks down starch into oligosaccharides through hydrolysis  Secreted by the humans parotid glands and the pancreas  -Amylase  -Amylase  -Amylase

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Factors that may affect catalysis rates

 Temperature  pH  Enzyme concentration  Amount of substrate

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Materials and Methods

Solution Preparation

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

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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.

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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.

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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.

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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.

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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.

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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

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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 (%) 0.5 0.75 1 1.5 2 2.5 time to achromic point (min) 30 0.833333333 23 1.630434783 18 2.777777778 11 6.818181818 7 14.28571429 7 17.85714286 0.0800 0.1000 0.1333 0.2000 0.2667 0.4000 amylase units Enzyme activity

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
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Effect of Enzyme Concentration

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

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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 (%) 0.5 0.75 1 1.5 2 2.5 time to achromic point (min) amylase units Enzyme activity

30 23 18 11 7 7

0.833333333 1.630434783 2.777777778 6.818181818 14.28571429 17.85714286

0.4000 0.2667 0.2000 0.1333 0.1000 0.0800

Figure1. Enzyme Activity with Varying % Concentrations of Salivary Amylase

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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)

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Effect of Enzyme Concentration

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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

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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

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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])

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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
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Michaelis-Menten Plot
0.000035 0.00003 Reaction Velocity ([Starch]/min) 0.000025 0.00002 0.000015 0.00001 0.000005 0 0 0.0002 0.0004 0.0006 [Starch] 0.0008 0.001 0.0012

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
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Michaelis-Menten Plot (Corrected)

0.0000205

Reaction Velocity ([Starch]/min)

0.00002

0.0000195

0.000019

0.0000185

0.000018 0 0.0002 0.0004 0.0006 [Starch] 0.0008 0.001 0.0012

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
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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)

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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)
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Lineweaver-Burk Plot
70000 1/ Reaction Velocity 60000 50000 40000 30000 20000 10000 0 0 1000 2000 1/ [Starch] 3000 4000 y = 11.411x + 23545 R = 0.7852

Double Reciprocal Plot

Not strong liinear relation (due to experimental limits) R2 value of only 0.785 Vmax= 4.24719E-05
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Km= 0.000484646

Lineweaver-Burk Plot (Corrected)

54500 54000 53500 1/ Reaction Velocity 53000 52500 52000 51500 51000 50500 50000 49500 0 1000 2000 3000 4000 5000 1/ [Starch] y = 2x + 48000 R = 1

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
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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

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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

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Effect of pH

Figure5. Effect of Varying pH in Enzymatic Activity of Amylase.

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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

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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

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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 5C and when frozen

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Effect of Temperature

Figure6. Effect of Varying temperature in Enzymatic activity.

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Effect of Temperature

highest enzyme activity was seen in the temperature 38C

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

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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.
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Thank you!!

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