US EPA

1399186 

Technical Report 

The effect of acrolein on L-asparaginase 2 from Escherichia coli 

Bilimoria MH, , JR; Nisbet MA, , JR 

1970 

HAPAB/71/01986 

Soc 

3 

HAPAB The effect of acrolein on L-asparaginase 2 from Escherichia coli was studied. The E. coli culture was grown by shaking on a rotary shaker at 100 rpm in a medium containing 1.0% peptone, 0.6% beef extract and 0.33% potassium phosphate (monobasic). A medium containing 2% Hycase and 0.02% yeast extract was also used. The partially purified preparation contained only about 3% L-asparaginase 1. Protein was determined spectrophotometrically using the method of Groves et al. (1968). The procedure and equations given by Campbell et al (1967) were used for determining the two forms of L-asparaginase. The enzyme and acrolein were incubated for varying lengths of time. The enzyme assays were carried out at pH 8.4 and 5.0 after the appropriate dilutions were made. The content of the preincubation mixtures was: 28 IU L-asparaginase 2, 50 and mcM acrolein and 25 mM borate (pH 8.4) or acetate (pH 5.0) At the appropriate time the mixtures were diluted 1:200 with physiological saline. L-asparaginase activity was then assayed in a mixture containing 0.2 mM borate or acetate buffer and 20 mcM L-asparaginase (Bilimoria, 1969). Of the sulfydryl reagents tested, only acrolein had an inhibitory effect (30% inhibition) on L-asparaginase 2 activity when used in a concentration of 50 mcM/ml of the preincubation mixture. The observed inhibition was more pronounced at the higher pH level studied (pH 8.4). The activity on the test enzyme was not inhibited by the -SH specific reagents, sodium arsenite and p-hydroxymercuribenzoate. 1970