Biochemistry calculations

A The influence of substrate concentrations on enzyme activity.

The following assays will be performed at room temp. (Amounts added are
in microlitres of each solution).

L-tyrosine 20 40 60 80 100

Tyrosinase 20 20 20 20 20

Phosphate buffer 860 840 820 800 780



The formation of the chromophore is measured at 475nm in the
spectrophotometer. After adding L-tyrosine, tyrosinase and buffer to
the cuvette the 3 are mixed and placed in the spectro. Followed by
passing the reference button to zero the instrument. Changes in
absorbance are measured after 5 minutes and 7 minutes. The change in
OD475 is determined at each substrate concentration by subtracting the
OD7min – OD5min = ∆OD475/2 min. Specific activities are calculated
for each determination as ∆OD475/mg of protein/min.

B The influence of enzyme concentration on activity

The following assays will be performed at room temperature.

Amounts added are in microlitres of each solution.

F

P

X

b

n

t

0

2

љ

°

ё

А

И

Р

Ш

40 50

Phosphate buffer 790 780 770 760 750



d ∆OD475 versus [E] enzyme.