Page 250 - The Manga Guide to Biochemistry

Enzyme Reaction Measurement

There are various methods of measuring enzyme activity, such as using radioisotopes to
measure the amount of a reaction product produced or using a change in color to measure
the reaction product formed when a substrate is acted on by an enzyme.

I will explain a method of using radioisotopes to measure the enzyme activity of DNA
polymerase and a method of using a color reaction to measure the enzyme activity of
α-amylase.

DNA Polymerase Activity Measurement Method

First, place the following in a mirco test tube: the solution for measuring activity (with pH
optimized and buffered for DNA polymerase), DNA polymerase, the DNA that is to be the
template, the nucleotide that is to be the raw material, and the magnesium chloride co-
factor. Add the nucleotide that contains the radioisotope, and let it react at 37° C for a fixed
interval.

When this is done, the nucleotides that contain the radioisotopes are captured in
the new DNA strands that have been synthesized by the DNA polymerase. The unreacted
nucleotides are removed, and the synthesized DNA strands are placed in a small radioiso-
tope bottle for measurement in a device called a liquid scintillation counter, which measures
radioisotopes. Since higher enzyme activity means more radioisotopes were captured in the
DNA, higher numeric values indicate higher enzyme activity.

Micro Radioisotope Radioisotopes
test tube (3H) are captured in
synthesized DNA.
Unreacted Sample is placed
nucleotides in the liquid
are removed. scintillation
counter.
DNA polymerase

Template DNA

nucleotide

DNA polymerase is reacted Small radioisotope Measurement
with nucleotides that measurement bottle
contain radioisotopes.

DNA polymerase activity
measurement method

236 Chapter 5