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.
