Date of Award

Spring 1990

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Thomas M Laue

Abstract

In the final step of coagulation in the horseshoe crab, coagulogen (M$\sb{\rm w}$ = 19,000, from sedimentation equilibrium) is converted by discrete proteolysis into its clottable form, coagulin. Coagulogen does not have a strong tendency to self-associate. In 0.1 M ammonium bicarbonate, 50 mM Tris-HCL (pH 8.1), trypsin generated coagulin rapidly associates to form a stable, solid gel. Polymerization is pH-dependent, with reversible depolymerization occurring in acid (midpoint pH 5.4). Sedimentation velocity analysis reveals that stable intermediates remain at pH 2.5, with 30-31 s being the predominant form along with monomer (1.89 s) and larger forms ($>$60 s). Near the midpoint of the titration, a pressure-dependent association occurs with a distribution of very large species resulting. There is also evidence that at this point a concurrent pressure-dependent depolymerization is occurring.

Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen in my preparations. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. A molecular weight of 16,300 was determined by analytical ultracentrifugation. This value is consistent with estimates from SDS-PAGE and amino acid composition. The amino-terminal sequence consists primarily of hydrophobic amino acid residues. LTI was found to inhibit the action of trypsin on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known trypsin inhibitors revealed no significant similarity to other trypsin inhibitors, suggesting it may represent a new class of proteinase inhibitors.

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