COMPARISON OF 9 DIFFERENT CASEINOLYTIC ASSAYS FOR ESTIMATION OF PROTEINASE ACTIVITY AND FURTHER IMPROVEMENT OF THE BEST METHOD

Comparative efficacies of nine different caseinolytic methods for assa y of proteinase activity indicated significantly different responses, the values being in the range of 34.10-192.30 units/g enzyme, even at constant ratio of enzyme to substrate and equal reaction time. The met hod of Nakanishi et al. estimated highest titres, i.e. 192.30 units/g enzyme at constant substrate to enzyme ratio and hence was selected fo r further improvement. Results indicated no change in estimation at 15 -60 min standing time of the reaction mixture at 30 degrees C, but the estimations increased by 10% at 60 degrees C standing temperature. Th e use of trichloro acetic acid mixture, to precipitate undigested prot ein and to simultaneously liberate tyrosine from the digestion product s, resulted in highest estimation, as compared to that with different concentrations of trichloro acetic acid. The methods used for estimati on of the digestion products were found to have greater impact, the me asurement as per the original procedure of Lowry et al. being most eff icient. The absorbance values for pure tyrosine by these methods were of linear nature and also showed highest value with the use of the ori ginal method of Lowry et al., thereby eliminating any role of interfer ing compounds in higher estimations. The presently formulated improved methodology led to 2.2 and 1.9 times increase in enzyme estimation, a s compared to generally followed Kunitz's method and the original meth od of Nakanishi et al., respectively.