Graduate Thesis Or Dissertation
 

Effect of environmental variables on the structure and function of hemocyanin from Callianassa californiensis (Decapoda: Thalassinidea)

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  • Oxygen binding behavior and subunit association-dissociation of the hemocyanin from Callianassa are sensitive to several environmental variables. Callianassa californiensis is a burrowing estuarine which must survive changes in salinity, temperature and oxygen availability. Previous work on the structure and function of the hemocyanin was done under conditions which did not duplicate physiological and environmental parameters. The present research was undertaken to define precisely what changes in oxygen binding behavior and subunit composition might occur under conditions the animal might reasonably be expected to encounter in its habitat. A series of buffered physiological saline solutions was developed which would mimic ionic conditions in the hemolymph at a given salinity and which would allow precise control of pH at a given temperature. These buffers were used for both oxygen binding and sedimentation velocity experiments. The first part of the thesis describes the results of oxygen binding measurements. The oxygen binding affinity of the 39S hemocyanin species drops only slightly when salinity drops from 35 o/oo to 17.5 o/oo and cooperativity is unaltered. The maximum Hill coefficient occurs at the physiological pH. The affinity of the 17S hemocyanin is similar, but the cooperativity is quite different. The second part of the thesis is concerned with structural changes in the hemocyanin. Callianassa hemocyanin exhibits a Mg²⁺ dependent reversible monomer-tetramer subunit association. Callianassa is an osmoconformer for all major ions. The effect of salinity, temperature and protein concentration on the amount of dissociation of subunits was determined. These three variables were related by an equation which describes a surface of equal dissociation, defined as an "isolytic" surface. By using computer graphics to visualize the surface it was determined that the equation predicts quite well the dissociation which can be observed in whole blood. The primary conclusion drawn from both lines of research is that the hemocyanin is remarkably stable, both structurally and functionally, and provides a kind of molecular homeostasis for an animal which must survive an unstable estuarine environment.
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