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

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 (K_i) of about 10 mM. (3) Both the esterolytic (AGLME hydrolysis) and the proteolytic activities (PK activation) were inhibited by diisopropylfluorosphosphate (DFP) with an approximate K_i of 5 × 10^–4 M.

• References

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