King Saud UniversityCollege of scienceBiochemistry department

Title of the experiment:

Trypsin activity

Objectives:

a) To determine trypsin activity by digestion of caseinb) To select the best casein concentration which will give optimum trypsin activity

Introduction:

The assay of the antitryptic activity depends mainly on the reaction between casein (substrate) and trypsin (enzyme) so it was necessary to determine the proper concentration of both the enzyme and substrate which will show the optimum proteolytic activity.

Expression of activity unit:

One trypsin unit is defined as the increase of 0.01 absorbance unit at 280 nm per 10 ml of the reaction mixture, under the set conditions.

The substrate concentration is one of the most important factors which determines the velocity of enzymes reactions. In most cases when initial velocity is plotted against substrate concentration a hyperbolic curve is obtained.

In the initial reaction keeping the enzyme concentration constant the rate of the reaction is proportional to the substrate concentration, so the reaction is a first order reaction with respect to the substrate.As the substrate concentration increases the initial rate increases less, so the reaction velocity is no longer proportional to the substrate concentration. In this region the reaction is a “mixed order” reaction.

With a further increase in the substrate concentration the reaction rate becomes independent of the substrate concentration and approaches a constant rate, here the reaction is a “zero order” reaction with respect to the substrate and the enzyme is saturated with the substrate and at this point maximum velocity occurs.

Method:

1. A series of dilutions were prepared from the casein stock solution by mixing casein with different volumes of buffer, as shown in table 1. The enzyme concentration was fixed at 0.015 mg/ml

2. The reaction started by adding 2 ml of trypsin to all tubes. The reaction will proceed for 20 min while incubated in a water bath at 37C and PH 7.6

3. In the blank tube T.C.A was added before the addition of the trypsin solution4. After 20 min the reaction is stopped by the addition of T.C.A to all tubes5. Tubes have to stand for one hour at room temperature and the precipitate was removed by

filtration6. Absorbance is read against the blank at 280 nm

Table 1

Solution A B C D E F GCasein2% w/v

0.4 0.8 1.2 1.4 1.8 2 1.4

Phosphate buffer

( pH 7.6)

1.6 1.2 0.8 0.6 0.2 - 0.6

Trypsin0.015 mg/ml

2 2 2 2 2 2 2

T C A5% w/v

6 6 6 6 6 6 6

Results:

Table 2

Tube Casein concentrationg/ml

Absorbance

A 0.004

B 0.008

C 0.012

D 0.014

E 0.018

F 0.02

A graph is to be plotted between the concentration of casein and absorbance at 280 nmKm and v max should be obtained from the graph