Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein

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Abstract
Synthetic procedures were developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2Cl and Pro-Phe-ArgCH2Cl, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. Pro-Phe-ArgCH2Cl inactivated plasma kallikrein 50% in 24 min at a concentration of 2 .times. 10-8 M, while other trypsin-like proteases were less susceptible in inactivation than killikrein, differing by a factor of 48 for plasmin and factors of 102-105 for factor Xa, thrombin and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2Cl (Ki = 0.078 .mu.M) was about 60 times that for Ala-Phe-LysCH2Cl (Ki = 4.9 .mu.M), whereas human plasmin exhibited about the same affinity for the former affinity label (Ki = 1.3 .mu.M) as for the latter (Ki = 0.83 .mu.M). The rate constants for the irreversible step of the affinity labeling reaction, K2, were similar for all affinity labels tested with the individual proteases: 0.35 min-1 for plasma killikrein and 0.18 min-1 for plasmin.

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