Activity of
Activity of Salivary Amy
Salivary Amylase
lase
Domingo, Guray, Hugo, Lorenzo, Mohammad Isa
Domingo, Guray, Hugo, Lorenzo, Mohammad Isa
Intro
Intro
Because everything has a start Because everything has a start
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Catalysis
Catalysis
The process of increasing the The process of increasing the rate of reaction with the use of rate of reaction with the use of a catalyst.a catalyst.
Catalyst – any substance that increases rate Catalyst – any substance that increases rate of reaction upon addition toof reaction upon addition to a certain reaction
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Enzymes
Enzymes
Act on substrates in a reactionAct on substrates in a reaction
Highly specificHighly specific
Breaks down complex macromolecules, synthesizes compoundsBreaks down complex macromolecules, synthesizes compounds essential for the cell
essential for the cell
Active siteActive site
Enzyme-substrate complexEnzyme-substrate complex
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http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg http://www.cas.muohio.edu/~wilsonkg/old/gene2005/syllabus_F03_23.jpg
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Enzymes
Enzymes
Require cofactors for activityRequire cofactors for activity
Classified according to the types of rClassified according to the types of reaction they catalyzeeaction they catalyze
–
– Oxidoreductase
Oxidoreductase
–
– Transferase
Transferase
–
– Hydrolase
Hydrolase
–
– Lyase
Lyase
–
– Isomerase
Isomerase
–
– Ligase
Ligase
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Amylase
Amylase
An enzyme that breaks down starch into oligosaccharides An enzyme that breaks down starch into oligosaccharides throughthrough hydrolysis
hydrolysis
Secreted by the human’s parotid glands and Secreted by the human’s parotid glands and the pancreasthe pancreas α-Amylaseα-Amylase β-Amylaseβ-Amylase γ-Amylaseγ-Amylase
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Factors that may affect catalysis rates
Factors that may affect catalysis rates
TemperatureTemperature
pHpH
Enzyme concentrationEnzyme concentration
Materials and Methods
Materials and Methods
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Solution Preparation
Solution Preparation
Saliva was collected.Saliva was collected.
1 ml of saliva was diluted to 1 ml of saliva was diluted to 10 ml with distilled water.10 ml with distilled water.
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Estimation of salivary amylase activity
Estimation of salivary amylase activity
A mixture of 5.0 mL 1% starch, A mixture of 5.0 mL 1% starch, 2 mL 1% NaCl solution and 2 mL 1% NaCl solution and 2 mL2 mL phosphate buffer put in a test
phosphate buffer put in a test tube and then placed in a tube and then placed in a water bathwater bath
At 38At 38ooC, 1 mL salivary enzyme solution added to C, 1 mL salivary enzyme solution added to the solution.the solution.
A drop from the digestion mixture mixed with 1 dA drop from the digestion mixture mixed with 1 drop of iodine for everyrop of iodine for every minute.
minute.
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Effect of
Effect of
enzyme concentration
enzyme concentration
The salivary The salivary amylase solution amylase solution diluted to diluted to five lower five lower concentrations: concentrations: 2.5%,2.5%, 2.0%, 1.5%, 1.0%, 0.75% and
2.0%, 1.5%, 1.0%, 0.75% and 0.5%.0.5%.
The same procedure done as previous using 1% concentration of starchThe same procedure done as previous using 1% concentration of starch solution.
solution.
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Effect of amount of substrate
Effect of amount of substrate
Six percent starch prepared from which five other dilutions wereSix percent starch prepared from which five other dilutions were prepared: 5%, 4%, 3%, 2%, and 1%.
prepared: 5%, 4%, 3%, 2%, and 1%.
The same procedure for Estimation of salivary amylase activity usedThe same procedure for Estimation of salivary amylase activity used using 2.5% salivary amylase solution.
using 2.5% salivary amylase solution.
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Effect of pH
Effect of pH
A mixture of 0.2 M sodium biphosphate (NaA mixture of 0.2 M sodium biphosphate (Na
2
2HPOHPO44) and 0.1 M citric acid) and 0.1 M citric acid
prepared to obtain different buffer solutions with pH varying from 3.0 prepared to obtain different buffer solutions with pH varying from 3.0 toto 8.0.
8.0.
Similarly, procedures from the estimation of enzymatic activity wereSimilarly, procedures from the estimation of enzymatic activity were applied, recording all notable reaction rates for
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Effect of temperature
Effect of temperature
Test tube with 2.5% salivary enzyme was placed on water bathsTest tube with 2.5% salivary enzyme was placed on water baths maintained at 4
maintained at 4ooC, 10C, 10ooC, 38C, 38ooC, 58C, 58ooC, 78C, 78ooC and 100C and 100ooC.C.
Results and Discussion
Results and Discussion
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Estimation of amylase activity
Estimation of amylase activity
Achromic point is the time it takes for Achromic point is the time it takes for
the enzyme to completely hydrolyze the the enzyme to completely hydrolyze the starch solution.
starch solution.
enzyme-starch mixture is not able toenzyme-starch mixture is not able to
produce a blue to violet color with produce a blue to violet color with iodine -> absence of starch
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Estimation of amylase activity
Estimation of amylase activity
Amylase units - amount of enzyme necessary to
Amylase units - amount of enzyme necessary to digest 5 ml 1% starch todigest 5 ml 1% starch to reach the achromic point within 10 minutes
reach the achromic point within 10 minutes Enzyme activity - mg starch hydrolyzed per minute per u
Enzyme activity - mg starch hydrolyzed per minute per unit enzymenit enzyme Effect of Enzyme Concentration
Effect of Enzyme Concentration Salivary
Salivary Amylase (%) Amylase (%)
time to achromic point time to achromic point
(min) (min)
a
ammyyllaasse e uunniittss EEnnzzyymmee activity activity 0.5 0.5 3030 0 0..88333333333333333 3 00..44000000 0.75 0.75 2323 1.630434783 1.630434783 0.26670.2667 1 1 1818 2 2..77777777777777778 8 00..22000000 1.5 1.5 1111 6 6..88118811881188118 8 00..11333333 2 2 77 1 144..228855771144229 9 00..11000000 2 2..55 77 17.85714286 17.85714286 0.08000.0800 Table1. Effect of
Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Enzyme Concentration on the Rate of Reaction. Different Dilutions of Saliva andSaliva and Their Corresponding Time to Reach the Achromic Points,
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Effect of Enzyme
Effect of Enzyme
Concentration
Concentration
enzyme + substrate <=> enzyme-substrate complex <=> enzyme + product enzyme + substrate <=> enzyme-substrate complex <=> enzyme + product
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Effect of Enzyme
Effect of Enzyme
Concentration
Concentration
Effect of Enzyme Concentration Effect of Enzyme Concentration Salivary Amylase
Salivary Amylase (%) (%)
ttimime e tto o aacchhrromomiic c ppooinint t ((miminn)) aammyylalasse e uunniitsts EEnnzzyymme e aaccttiviviittyy
0.5 0.5 3030 0.833333333 0.833333333 0.40000.4000 0.75 0.75 2323 1.630434783 1.630434783 0.26670.2667 1 1 1818 2.777777778 2.777777778 0.20000.2000 1.5 1.5 1111 6 6..88118811881188118 8 00..11333333 2 2 77 1 144..228855771144229 9 00..11000000 2.5 2.5 77 17.85714286 17.85714286 0.08000.0800 Table1. Effect of
Table1. Effect of Enzyme Concentration on the Rate of Reaction. Different Dilutions of SEnzyme Concentration on the Rate of Reaction. Different Dilutions of S aliva andaliva and Their Corresponding Time to Reach the Achromic Points, A
Their Corresponding Time to Reach the Achromic Points, A mylase Units and Enzyme Activitymylase Units and Enzyme Activity
Figure1. Enzyme Activity with Varying % Concentrations
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Effect of Enzyme
Effect of Enzyme
Concentration
Concentration
Increased enzyme concentration increases reaction rate (more en
Increased enzyme concentration increases reaction rate (more enzymeszymes are present to act upon
are present to act upon a fixed amount of substrate)a fixed amount of substrate)
However, as substrate concentration is constant, it produces a
However, as substrate concentration is constant, it produces a limitinglimiting effect
effect on reaction ron reaction rate (excess enate (excess enzymes begin zymes begin to compete to compete for substrate)for substrate)
Surplus of enzymes on a
Surplus of enzymes on a limited reaction rate causes overall enzymelimited reaction rate causes overall enzyme activity to diminish (reaction rate cannot cope up
activity to diminish (reaction rate cannot cope up with increased enzymewith increased enzyme conc)
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Effect of Enzyme
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Effect of the Amount of Substrate
Effect of the Amount of Substrate
Rate of ReactionRate of Reaction –
– describes how fast a chemical reaction proceedsdescribes how fast a chemical reaction proceeds –
– depends on reactant and product concentrationsdepends on reactant and product concentrations •• More importantly on rate constant kMore importantly on rate constant k
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Effect of the Amount of Substrate
Effect of the Amount of Substrate
Enzyme KineticsEnzyme Kinetics –
– still follows the same trendstill follows the same trend
•• Increasing either substrate or enzyme increases rateIncreasing either substrate or enzyme increases rate –
– but but there is there is a limit a limit to this to this relationrelation
•• When enzyme When enzyme conc are constant, conc are constant, there is a limit there is a limit to the veloto the velocity of thecity of the reaction
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Michaelis-Menten KineticsMichaelis-Menten Kinetics –
– enzymatic reactions are observed to reach a maximum rate of enzymatic reactions are observed to reach a maximum rate of reaction Vreaction Vmaxmax –
– constant enzyme concentration provides a limiting effectconstant enzyme concentration provides a limiting effect •• All enzymes are bound to substrateAll enzymes are bound to substrate
– – v v
o
o= V= Vmaxmax [S] / (K[S] / (Kmm+ [S])+ [S])
Effect of the Amount of Substrate
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Rectangular Hyperbola
Rectangular Hyperbola
Max rate of Vmax
Max rate of Vmax
Half-Velocity is reached
Half-Velocity is reached
at Km
at Km
Vmax is dependent on
Vmax is dependent on
[E}
[E}
Km is constant
Km is constant
–
– Km=K
Km=K
-1-1+ K
+ K
22/ K
/ K
+1+1..
–
– Measure of affinity
Measure of affinity
Effect of the Amount of Substrate
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Rate of Reaction vs. Substrate Concentration
Rate of Reaction vs. Substrate Concentration
Rate increases with substrate concentration Rate increases with substrate concentration However exponential relation
However exponential relation
Due to experiment limit (30 min) Due to experiment limit (30 min) Km and Vmax not evident
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Rate of Reaction vs. Substrate Concentration
Rate of Reaction vs. Substrate Concentration
Allowing time to go beyond 30, rectangular hyperbola is
Allowing time to go beyond 30, rectangular hyperbola is attainedattained Change in
Change in rate diminishes rate diminishes as substrate as substrate conc increasesconc increases Vmax still indiscernible together with Km
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Asymptotical nature makes it hard to determine which much certainty theAsymptotical nature makes it hard to determine which much certainty the values of Km and Vmax
values of Km and Vmax
Algebraic manipulation (double-reciprocal plot) allows linear expression of Algebraic manipulation (double-reciprocal plot) allows linear expression of MM eq.
MM eq.
Lineweaver-Burke EquationLineweaver-Burke Equation –
– : 1/: 1/v v
o
o = (Km / V= (Km / Vmaxmax) (1/ [S]) + (1/V) (1/ [S]) + (1/Vmaxmax))
Effect of the Amount of Substrate
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Linear equation
Linear equation
Regression Analysis
Regression Analysis
allows determination of
allows determination of
Km and Vmax
Km and Vmax
Also able to determine
Also able to determine
nature of protein function
nature of protein function
inhibition (competitive,
inhibition (competitive,
uncompetitive,
uncompetitive,
noncompetitive)
noncompetitive)
Effect of the Amount of Substrate
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Double Reciprocal Plot
Double Reciprocal Plot
Not strong liinear relation (due
Not strong liinear relation (due to experimental limits)to experimental limits) R2 value of only 0.785
R2 value of only 0.785 Vmax=
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Perfect linear relation Perfect linear relation Vmax=
Vmax= 2.08333E-05 2.08333E-05 Km=4.16667E-05Km=4.16667E-05 Very Low Km, high affinity of enzyme
Very Low Km, high affinity of enzyme Vmax close to velocity values, near sa
Vmax close to velocity values, near sa turationturation
Double Reciprocal Plot
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Effect of pH
Effect of pH
•• Enzymes are proteinsEnzymes are proteins
–
– function is ultimately determined by their structurefunction is ultimately determined by their structure
•• optimal pH rangeoptimal pH range
–
– Changes in pHChanges in pH
•• excess of either Hexcess of either H++ or OHor OH-- ionsions
•• affect the secondary, tertiary and quarternary structures affect the secondary, tertiary and quarternary structures by disruptingby disrupting hydrogen bonds and van der wal interactions.
hydrogen bonds and van der wal interactions. •• change the active site of the enzymeschange the active site of the enzymes
–
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Effect of pH
Effect of pH
•• EnzymesEnzymes –– have a range of pH at which it is have a range of pH at which it is active and outside of which it is inertactive and outside of which it is inert –
– optimum pHoptimum pH
•• most favorable pH valuemost favorable pH value
•• point where the enzyme is most activepoint where the enzyme is most active
•• extremely high or low pH values generally result in complete loss of activityextremely high or low pH values generally result in complete loss of activity for most enzymes
for most enzymes
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Effect of pH
Effect of pH
Figure5. Effect of Varying pH
Figure5. Effect of Varying pH in Enzymatic Activity of in Enzymatic Activity of Amylase.
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Effect of pH
Effect of pH
enzymatic activity of salivary amylase is highest at pH 7enzymatic activity of salivary amylase is highest at pH 7 –
– pH of oral cavity is close to 7pH of oral cavity is close to 7
At pH 8,At pH 8, –
– decrease in activitydecrease in activity
acidic pH 3, 4 and 5acidic pH 3, 4 and 5 –
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Effect of Temperature
Effect of Temperature
•• reaction rate of an enzymatic reaction increases reaction rate of an enzymatic reaction increases as the temperature isas the temperature is raised
raised
•• 10° C rise in 10° C rise in temperature will increase the activity of most enzymes by 50temperature will increase the activity of most enzymes by 50 to 100%
to 100%
•• many enzymes are adversely affected by high temperaturesmany enzymes are adversely affected by high temperatures
–
– Reaction rates may increase with temperature up to a maximum level, but thenReaction rates may increase with temperature up to a maximum level, but then abruptly decline with further increase of temperature
abruptly decline with further increase of temperature
•• increases in temperature are able to break H-bonds and van der walincreases in temperature are able to break H-bonds and van der wal interactions
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Effect of Temperature
Effect of Temperature
decrease in temperature, rate of reaction is decrease in temperature, rate of reaction is decreased due to lowereddecreased due to lowered energy
energy
over a period of time, enzymes will be dover a period of time, enzymes will be deactivated at even moderateeactivated at even moderate temperatures
temperatures
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Effect of Temperature
Effect of Temperature
Figure6. Effect of Varying temperature
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Effect of Temperature
Effect of Temperature
•• highest enzyme activity was seen in the temperature 38°Chighest enzyme activity was seen in the temperature 38°C
–
– near temperature in the oral cavitynear temperature in the oral cavity
•• other temperature levelsother temperature levels
–
– enzyme activity was found to be enzyme activity was found to be minimalminimal –
– achromic point was not rachromic point was not reached within the 30min limiteached within the 30min limit
•• optimum enzyme activity is at that level closest to the optimum enzyme activity is at that level closest to the naturalnatural physiological setting
physiological setting •• high heathigh heat
–
– denaturationdenaturation
•• very low temperaturesvery low temperatures
–
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Conclusion
Conclusion
1 ml of human saliva has 1 ml of human saliva has around 17.86 Units of amylase.around 17.86 Units of amylase.
Increased enzyme concentration increases reaction rate, however, itIncreased enzyme concentration increases reaction rate, however, it decreases overall enzyme activity.
decreases overall enzyme activity.
Increased substrate concentration increases reaction rate, however thereIncreased substrate concentration increases reaction rate, however there is a maximum rate that can be
is a maximum rate that can be achieved. (Vmax)achieved. (Vmax)
Michaelis-Menten and Lineweaver-Burke describes enzyme kinetics. TheMichaelis-Menten and Lineweaver-Burke describes enzyme kinetics. The Michaelis constant Km gives an idea on enzyme affinity.
Michaelis constant Km gives an idea on enzyme affinity.
Salivary amylase has a Km = 4.84e-4 Salivary amylase has a Km = 4.84e-4 (corrected: 4.167e-5)(corrected: 4.167e-5)
There is an optimal pH and There is an optimal pH and temperature range for enzyme activity.temperature range for enzyme activity. Outside this range, enzyme activity drastically decreases due to Outside this range, enzyme activity drastically decreases due to denaturation and deactivation.
denaturation and deactivation.
Optimal pH would be near 7 while optimal temperature should be Optimal pH would be near 7 while optimal temperature should be near 37near 37 C.
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