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 zero- order 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.