salivary amylase
TRANSCRIPT
Activity of Salivary Amylase
Domingo, Guray, Hugo, Lorenzo, Mohammad Isa
IntroBecause everything has a start
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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 human’s parotid glands and the pancreas
α-Amylase
β-Amylase
γ-Amylase
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Factors that may affect catalysis rates
Temperature
pH
Enzyme concentration
Amount of substrate
Materials and Methods
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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.
Results and Discussion
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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
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
Effect of Enzyme Concentration
Salivary Amylase (%)
time to achromic point (min)
amylase units Enzyme activity
0.5 300.833333333 0.4000
0.75 231.630434783 0.2667
1 182.777777778 0.2000
1.5 116.818181818 0.1333
2 714.28571429 0.1000
2.5 717.85714286 0.0800
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
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Effect of Enzyme Concentration
enzyme + substrate <=> enzyme-substrate complex <=> enzyme + product
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Effect of Enzyme Concentration
Effect of Enzyme Concentration
Salivary Amylase (%)
time to achromic point (min) amylase units Enzyme activity
0.5 30 0.833333333 0.4000
0.75 23 1.630434783 0.2667
1 18 2.777777778 0.2000
1.5 11 6.818181818 0.1333
2 7 14.28571429 0.1000
2.5 7 17.85714286 0.0800
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
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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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])
Effect of the Amount of Substrate
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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
Effect of the Amount of Substrate
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Rate of Reaction vs. Substrate ConcentrationRate increases with substrate concentration
However exponential relation
Due to experiment limit (30 min)
Km and Vmax not evident
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Rate of Reaction vs. Substrate ConcentrationAllowing 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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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)
Effect of the Amount of Substrate
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Linear equation
Regression Analysis allows determination of Km and Vmax
Also able to determine nature of protein function inhibition (competitive, uncompetitive, noncompetitive)
Effect of the Amount of Substrate
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Double Reciprocal PlotNot strong liinear relation (due to experimental limits)
R2 value of only 0.785
Vmax= 4.24719E-05 Km= 0.000484646
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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
Double Reciprocal Plot
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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 5°C 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 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
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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!!