Introduction to Enzyme and Coenzyme Chemistry - E-Library Home

52 Chapter 4
(1) Direct UV
(2) Radiochemical
S
ENZ
P (UV active at 394 nm)
ENZ
S* P*
separate P*
A 394
+ ENZ
slope v
P*
(counts per
minute)
scintillation
counting
time
time
(3) Indirect UV
S
ENZ
P
NADH NAD +
A 340
+ ENZ
slope v
UV active at 340 nm
time
(4) Coupled UV assay
S
ENZ
P
ENZ 2
Q
NADH NAD +
excess of coupling enzyme
monitor decrease in absorbance at 340 nm
Figure 4.1 Types of enzyme assays. A 340 , ultraviolet (UV) absorbance at 340 nm; A 394 , UV absorbance
at 394 nm; ENZ, enzyme; NADH, nicotinamide adenine dinucleotide, P, product; Q, product
of coupling enzyme; S, substrate.
a micro-organism. Enzymes are generally produced in the cytoplasm contained
within the cells of the producing organism, so in order to isolate the enzyme we
must break open the cells to release the enzymes inside (Figure 4.2). If it is a
bacterial source, the bacteria can be grown in culture media and the cells
harvested by centrifugation. The bacterial cell walls are then broken by treat-
enzymes
Obtain or grow cells
containing enzyme
cell wall
lyse cells at high pressure
Figure 4.2 Preparation of an enzyme extract.
extract
cell
debris
high speed
centrifugation