Download 2. Enzyme activity - Lectures For UG-5

MEASUREMENT OF ENZYME
ACTIVITY
UG-3rd
Recall……
1. Reaction rate: is defined as the amount of reactant modified per
unit time.
Units: μmoles/sec or mol/min
2. Enzyme activity:
is the ability of an enzyme to modify a
reactant.
1 unit (U) is the enzyme activity that converts 1
μmole of reactant per min under standard conditions.
Measurement of Enzyme Activity
1. It is a convenient method of enzyme quantitation.
2. The activity is related to concentration .
3. Common methods might photometrically measure :
• an increase In product concentration.
•
•
•
a decrease in substrate concentration.
a decrease in coenzyme concentration.
an increase in the concentration of an altered coenzyme.
zero-order kinetics
• Enzymes concentrations are always performed in zeroorder kinetics with substrate in sufficient excess to ensure
that not more than 20% of the available substrate is
converted to product.
• Any coenzymes also must be in excess.
• NAD or NADH is often convenient as a reagent for a
coupled- enzyme assay.
• In other coupled-enzyme assays, more than one enzyme
is added in excess as a reagent and multiple reactions are
catalyzed .
When performing an enzyme quantitation in zero-order
kinetics:
• Inhibitors must be lacking and other variables that may influence the
rate of the reaction must be carefully controlled.
•A constant pH should be maintained
The temperature should be constant within
0.1  C throughout the assay at a temperature
at witch the enzyme is active (25 C,30 C,or
37 C)
• The period for the analysis also must be carefully selected
• Enzyme quantitations must be performed during the linear
phase of the reaction.
Note:
When the enzyme initially introduced to the reactants and the excess
substrate is steadily combining with available enzyme, the reaction rate rises.
After the enzyme is saturated, the rates of product formation, release of
enzyme ,and recombination with more substrate proceed linearly. 6to8
minutes after reaction initiation ,the rate decreases as the substrate is
depleted, the reverse reaction is occurring appreciably and the product begins
to inhibit the reaction.
Two general methods used to measure the extent
of an enzymatic reaction:
1. FIXED-TIME:
The reactants are combined
The reaction proceeds for a designated time
The reaction is stopped (with weak acid)
The measurement is made of the amount of
reaction that has occurred
The larger the reaction,the more enzyme is present.
2. CONTINOUS-MONITORING OR KINETIC ASSAYS:
Multiple measurements ,usually of absorbance change ,are
made during the reaction ,either at specific time
intervals(usually every 30 or 60seconds)
Or continuously by a continuous-recording spectrometer.
Continuous measurements are preferred because the linearity of
the reaction more adequately verified and any deviation from
linearity is readily observable.
Enzyme activity
measurements may not be
accurate if:
Storage conditions compromise
integrity of the protein.
Enzyme inhibitors are present.
Or if necessary cofactors are not
present.