It has been noted in the literature that kallikrein has activity on synthetic substrates4"5. We have used these substrates to differentiate trypsin from kallikrein. The compound benzoyl arginine ethyl ester (BAEE) is a specific substrate for trypsin-like enzymes. Hydrolysis of BAEE yields benzoyl arginine and ethyl alcohol and can be measured directly by following the increase in optical density at 253 mpi on a Beckman DU spectrophotometer. This increase is directly proportional to the amount of enzyme present6. Utilizing 10~3 M BAEE in 0-05 M phosphate buffer pH. 7-0 we have derived activity curves for kallikrein (Fig. 1). Fig. 1.Indicated amounts of enzyme incubated with 3-0 ml. 10~8 M BAEE in total volume of 3-4 ml.Fig. 2. Relationship of BAEE concentration to reaction velocity. Km = 3-7 x 10-4 Fig. 3.Relationship of B AME concentration to reaction velocity. Km = 1-8 x 10-4By varying the substrate concentration, it is possible to deduce Michaelis constants for kallikrein. Using the equations of Lineweaver and Burke7: ~ = -^- -f -^ o>^ v V V we have found the Km of kallikrein on BAEE to be 3-7 x 10-4 (Fig. 2).We have also measured the Km of kallikrein for benzoyl arginine methyl ester and find it to be 1-8 x 10-(BAME) (Fig. 3). Both Km's differ greatly from the Km of trypsin, which is 8-0 x 10~5 (ref. 8). The similarity of the two m's for kallikrein is to be expected since trie stereochemistry of the two substrates BAEE and BAME is similar. Addition of 10~2 M calcium chloride results in a 1-25 increase in activity of trypsin as reported by Neurath9. We have been able to show the increase in activity with respect to trypsin but 10~2 M Cahas no stimulatory effect on kallikrein. The synthetic ester toluene sulphonyl arginine methyl ester (TAME) is active with trypsin and its activity can be measured by increase in optical density of 253 m[jt. Kallikrein has no activity with TAME (Table 1). To rule out the contamination of kallikrein by chymotrypsin, the enzyme was assayed for chymotryptic activity with the synthetic substrate acetyl tyrosine ethyl ester (ATEE) by the method of Schwert and Takenaka6. No activity for chymotrypsin was detected.Table 1. COMPARISON OF KALLIKREIN AND TRYPSIN ON TAME J O.D. with TAME x 10-3/h0 2,600Enzyme 200 ng Kallikrein 20 //g TrypsinA O.D. with BAEE x 10-3/h 2,70010,000 Kallikrein was found to lyse fibrin clots by the method of Astrup and Mullertz and Lassen10 and also to have caseinolytic activity. No plasminogen activating activity could be determined. Thus, kallikrein is unable to convert plasminogen to plasmin. (We would like to thank Dr. Edwin O. Salzman, who performed these assays.)The enzyme loses its activity above 55 C. It shows the same pH. optimal curve as reported by Webster5. Differences in the action of trypsin inhibitors on kallikrein and trypsin were also found. These differences will be the subject of another publication. This evidence strongly suggests that kallikrein is a distinct enzyme. Further evidence must await greater purification of kallikrein.We thank Dr. G. Cronheim, Hiker Laboratories, North-ridge, California, who supplied the partially purified hog pancreatic kallikrein (1 Frey unit/88 pig). This work was supported by the U.S. Public Health Service, grant No. ^.-5900.