Date of Award

Winter 1994

Project Type

Dissertation

Program or Major

Biochemistry

Degree Name

Doctor of Philosophy

First Advisor

Thomas M Laue

Abstract

The blood of the horseshoe crab Limulus polyphemus gels upon contact with gram negative bacterial endotoxin. Such contact induces the discrete proteolytic cleavage of a single protein, coagulogen. The product, coagulin, then assembles into a highly turbid gel. An investigation of the gelation and the physical properties of coagulogen and coagulin is described here. Except where noted, polymerization was induced by limited trypsinolysis of coagulogen.

Analytical centrifugation demonstrates that coagulogen only weakly self-associates. Fluorescence quenching indicates that the single tryptophan of coagulogen lies near the surface of the protein. The effective charge of coagulogen at pH 8, measured by equilibrium electrophoresis, is +1.0.

Electron microscopy reveals the gel to consist of a network of long, branching, intermeshed fibers about 10 to 20 nanometers wide. Centrifugation studies show that coagulin gels depolymerize to monomeric coagulin in 6 molar guanidine hydrochloride, but not in other commonly used denaturants. Gelation follows the removal of guanidine hydrochloride. Guanidine hydrochloride has modest effects on the fluorescence or sedimentation of coagulogen. Thus, depolymerization probably does not require gross structural changes in coagulin. It appears that a combination of ionic interactions, hydrogen bonding and hydrophobic interactions holds coagulin monomers together in the gel, with no one type of interaction dominating gel stability.

During gelation, changes in turbidity lag changes in the wavelength dependence, suggesting a sudden appearance of the gel phase. At sufficient concentration, coagulin monomers may form protofibrils that laterally associate into branching, interlacing fibers, creating the gel. An apparent monomer-gel equilibrium follows. At fifteen degrees celsius, an apparent first order rate constant of 0.055 per minute was found for the association of monomer with the gel. The critical concentration at that temperature was too small to measure.

During gelation, neither coagulogen, nor at least one of two expected intermediates leading to coagulin, participates in gelation. Reactions of coagulogen with chymotrypsin suggest that the other intermediate can gel, and suggests that no part of coagulogen need be freed for gelation to proceed. Thus, a decrease in osmotic pressure could accompany chymotrypsin-induced gelation. The increase in entropy that appears to drive gelation would have to overcome this osmotic effect.

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