King Saud University College of science Biochemistry department

Title of the experiment: Trypsin activity Objectives: a b To determine trypsin activity by digestion of casein To select the best casein concentration !hich !ill give optimum trypsin activity

"ntroduction: The assay of the antitryptic activity depends mainly on the reaction bet!een casein #substrate and trypsin #en$yme so it !as necessary to determine the proper concentration of both the en$yme and substrate !hich !ill sho! the optimum proteolytic activity% &xpression of activity unit: One trypsin unit is defined as the increase of '%'( absorbance unit at )*' nm per (' ml of the reaction mixture+ under the set conditions% The substrate concentration is one of the most important factors !hich determines the velocity of en$ymes reactions% "n most cases !hen initial velocity is plotted against substrate concentration a hyperbolic curve is obtained% "n the initial reaction ,eeping the en$yme concentration constant the rate of the reaction is proportional to the substrate concentration+ so the reaction is a first order reaction !ith respect to the substrate% -s the substrate concentration increases the initial rate increases less+ so the reaction velocity is no longer proportional to the substrate concentration% "n this region the reaction is a .mixed order/ reaction% 0ith 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 .$ero order/ reaction !ith respect to the substrate and the en$yme is saturated !ith the substrate and at this point maximum velocity occurs%

1ethod: (% - series of dilutions !ere prepared from the casein stoc, solution by mixing casein !ith different volumes of buffer+ as sho!n in table (% The en$yme concentration !as fixed at '%'(2 mg3ml )% The reaction started by adding ) ml of trypsin to all tubes% The reaction !ill proceed for )' min !hile incubated in a !ater bath at 45C and 67 5%8 4% "n the blan, tube T%C%- !as added before the addition of the trypsin solution 9% -fter )' min the reaction is stopped by the addition of T%C%- to all tubes 2% Tubes have to stand for one hour at room temperature and the precipitate !as removed by filtration 8% -bsorbance is read against the blan, at )*' nm Table (
Solution Casein )= !3v 6hosphate buffer # p7 5%8 Trypsin '%'(2 mg3ml TC2= !3v '%9 (%8 ) 8 B '%* (%) ) 8 C (%) '%* ) 8 : (%9 '%8 ) 8 & (%* '%) ) 8 ; ) > ) 8 < (%9 '%8 ) 8

?esults: Table )
Tube B C : & ; Casein concentration g3ml '%''9 '%''* '%'() '%'(9 '%'(* '%') -bsorbance

- graph is to be plotted bet!een the concentration of casein and absorbance at )*' nm Km and v max should be obtained from the graph