BBCCL-108 ENZYMES
EXPERIMENT 3
EFFECT OF pH ON ENZYME
ACTIVITY
Structure
3.1 Introduction 3.5 Effect of pH on Activity of
acid Phosphatase - Protocol
Expected Learning Outcomes
3.6 Precautions
3.2 Principle
3.7 Summary
3.3 Reagents
3.4 Preparation of Enzyme
Extract
3.1 INTRODUCTION
You have already learned in Experiment 1 the basics of assay of activity of an
enzyme taking acid phosphatase as an example. You also know that most of
the enzymes are proteins and amino acids are the fundamental units of all
proteins. Moreover, the amino acid composition and sequence of amino acid
residues determines the properties of proteins (refer Course BBCCT-101 Unit
2 & 3). Let us think, what will be the effect if the pH of medium surrounding
the protein molecules is changed? The integrity of native conformation of
protein is very essential for its biological function. Understandably, the
primary, secondary, tertiary and quaternary structures are very essential for
catalytic activity of enzymes. The catalytic activity of an enzyme is tightly
linked to the specific shape and chemical properties of its active site.
Changing the pH affects the V (velocity of enzyme catalyzed reaction), Km
(Michaelis constant), or the stability of the enzyme protein. To explain further,
the change in pH of medium can change the ionization of various groups of
the enzyme molecule which may alter the affinity of the enzyme for its
substrate. Ionization of the substrate may also be affected which can influence
the binding of the substrate to the enzyme. Extremes of pH may affect the
protein structure/stability.
Expected Learning Outcomes
After studying this unit, you should be able to:
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Experiment 3 Effect of pH on Enzyme Activity
understand the principle of effect of pH of assay mixture on activity of an
enzyme; and
perform the experiment to determine the effect of pH on activity of acid
phosphatase and understand the pH-activity profile.
3.2 PRINCIPLE
Most enzymes are proteins and are affected by change in pH of medium. The
pH at which activity of an enzyme is maximal is known as its optimum pH.
The activity of the enzyme decreases at pH higher or lower than the optimum
pH. At extremely high or low pH the active site of enzyme can be badly
affected, and generally a complete loss of enzyme activity is observed.
Optimum pH for acid phosphatase activity can be determined by assay of
enzyme activity in the acidic range e.g. using citrate buffer ranging from pH 3.4
to 6.2. The enzyme catalyses the conversion of substrate, p-nitrophenyl
phosphate to p-nitrophenol and inorganic phosphate (Pi). The optimum pH can
be shown by plotting a graph, pH vs. activity.
Note:-
1. For the determination of optimum pH/maximal activity, pH vs.
Absorbance410nm will be plotted.
2. Reactions for substrate conversion etc are given in Expt. 1.
3.3 REAGENTS
(i) Buffers: 0.1M Sodium citrate buffer ( pH 3.4 to 6.2)
Solution A: 0.1 M citric acid monohydrate (C6H8O7.H2O MW 210.14). Dissolve
21.014 g/liter in double distilled water
Solution B: 0.1M trisodium citrate dihydrate (C6H5O7Na3.2H2O MW 294.12).
Dissolve 29.412 g/liter in double distilled water
Mix solution A and B as given below Table 3.1:
Table 3.1: Preparation of 0.1 M Sodium Citrate Buffer
pH 3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2
Solution A 73.0 63.5 54.0 44.5 35.0 25.5 16.0 8.0
(mL)
Solution B 27.0 36.5 46.0 55.5 65.0 74.5 84.0 92.0
(mL)
(ii) Substrate solution: p-nitrophenyl phosphate (pNPP), 15 mM dissolved in
double distilled water/ different citrate buffers ranging from pH 3.0 – 6.2.
(iii) 0.2N Sodium hydroxide solution
Dissolve 8 gm sodium hydroxide pellets in 1000 mL distilled water.
(Standardize with 0.1N oxalic acid using phenolphthalein as indicator).
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BBCCL-108 ENZYMES
3.4 PREPARATION OF ENZYME EXTRACT
Follow the steps as already explained in Ex. 1 or 2 in this unit.
3.5 EFFECT OF pH ON ACTIVITY OF ACID
PHOSPHATASE-PROTOCOL
Pipette out the following reagents (Table 3.2) into clean test tubes in duplicate.
Prepare another set also and mark as Blank e.g. B1, B2, B3, B4 B5, B6, B7,
B8 corresponding to each pH.
Table 3.2: Preparation of Substrate solutions (at different pH).
-------------------------------- pH -----------------------------------------------
-
Reagent
3.4 3.8 4.2 4.6 5.0 5.4 5.8 6.2
Buffer (mL) 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.4
15 mM 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
pNPP
solution
(mL)
o
Mix properly and equilibrate at 37 C for ~3 min
Start the reaction by adding 0.1 mL of enzyme extract in each tube (Test
only). Note down the time of adding the enzyme extract in first tube.
Immediately mix properly and incubate at 37 o C for 10 min.
Stop reaction by adding 4.0 mL 0.2N NaOH in each tube.
In blanks (B1-B8), add enzyme extract (0.1 mL in each tube) at the end
after addition of 0.2N NaOH.
Record the absorbance of each tube (Test) against the corresponding
blank (prepared for each pH), at 410 nm using a spectrophotometer.
Plot the absorbance vs. pH, and find the peak value of absorbance and
the corresponding pH.
The pH value at which maximum absorbance is recorded is the optimum
pH for the acid phosphatase in given sample.
Note: i) If the absorbance at 410 nm (A410) in test sample is too high, dilute
the enzyme extract appropriately and repeat the experiment. 17
Experiment 3 Effect of pH on Enzyme Activity
ii) If the color developed (A410) is insufficient for 10 minutes, increase the
incubation time.
iii) The activity of enzyme (units/mL) can also be calculated by finding the
amount of para-nitrophenol produced (micromoles/min) from standard curve or
Extinction coefficient (ε).
3.6 PRECAUTIONS
i) Always wear gloves while working with p-nitrophenylphosphate and p-
nitrophenol. Avoid contact, inhalation etc.
ii) Ensure equilibration at incubation temperature before adding the enzyme.
iii) Blank is required for each pH.
3.7 SUMMARY
Acid phosphatase acts to liberate phosphate under acidic conditions and is
made in the liver, spleen, bone marrow and prostate gland. The effect of pH
on enzyme activity can be determined by using the synthetic substrate, p-
nitrophenylphosphate (at different pH in acidic range) and recording the
absorbance due to the formation of p-nitrophenol (measured as p-
nitrophenolate under defined conditions), spectrophotometrically at 410 nm.
The pH at which maximum absorbance/activity is observed is the optimum pH
for an enzyme, here acid phosphatase.
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