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Thromb Haemost 1974; 31(01): 030-039
DOI: 10.1055/s-0038-1649143
Original Article
Schattauer GmbH

A Direct Enzymatic Assay for the Esterolytic Activity of Activated Hageman Factor

Richard J Ulevitch
1   Department of Experimental Pathology, Scripps Clinic and Research Foundation, 476 Prospect Street, La Jolla, California 92037
,
David Letchford
1   Department of Experimental Pathology, Scripps Clinic and Research Foundation, 476 Prospect Street, La Jolla, California 92037
,
C. G Cochrane
1   Department of Experimental Pathology, Scripps Clinic and Research Foundation, 476 Prospect Street, La Jolla, California 92037
› Author Affiliations
Further Information

Publication History

Received 21 August 1973

Accepted 11 December 1973

Publication Date:
29 June 2018 (online)

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Summary

A direct enzymatic assay for activated Hageman factor (HFa) has been developed utilizing N-α-acetylgiycine lysine methyl ester (AGLME). This assay allowed the measurement of the esterolytic activity of purified Hageman factor (HF) which had been activated by enzymatic (trypsin) and non-enzymatic (kaolin) mechanisms. No esterolytic activity was detected in preparations of purified HF prior to activation.

Studies with purified HF activated on kaolin demonstrated the following: (1) Simple saturation kinetics (Km = 9 mM, Turnover number = 2.5 min–1) at AGLME concentrations less than 0.04 M and substrate inhibition at AGLME concentrations greater than 0.04 M. (2) The substrate AGLME behaved as a competitive inhibitor of the activation of prekallikrein (PK) by HFa with an inhibition constant (Ki) of about 10 mM. (3) Both the esterolytic (AGLME hydrolysis) and the proteolytic activities (PK activation) were inhibited by diisopropylfluorosphosphate (DFP) with an approximate Ki of 5 × 10–4 M.

 

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