Date of Award:

5-1983

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Chemistry and Biochemistry

Advisor/Chair:

Thomas M. Farley

Abstract

Nine hemoglobins have been isolated from the blood of cutthroat trout. All nine hemoglobins bind oxygen cooperatively and appear to be tetramers with molecular weights of -64,000. The oxygen equilibria and subunit structures of the purified hemoglobins were studied. In addition, the red blood cells of cutthroat trout were examined for the presence of ATP and GTP, which are known to be physiological modulators of hemoglobins in fishes.

Five hemoglobins with isoelectric points from 9.1 to 7.0 are classified as cathodal hemoglobins. These five hemoglobins have identical oxygen binding properties by the criteria tested. All have oxygen equilibria which are unaffected by protons and ATP and essentially independent of temperature, with overall enthalpies of oxygenation ~0. Two hemoglobins with isoelectric points near 6.5, classified as a nodal hemoglobins, have oxygen binding properties distinctly different from those of the cathodal hemoglobins. Both are characterized by a Root effect, displaying non-cooperative oxygen binding and low oxygen affinity at pH 6.2. ATP causes a large reduction in the oxygen affinity without affecting the cooperativity of oxygen binding. GTP has a similar but slightly larger effect on both hemoglobins. The oxygen equilibria of the anodal hemoglobins are temperature dependent, with the oxygen affinity being reduced as temperature increases. The overall enthalpy of oxygenation is -14 kcal/mol for both hemoglobins. The two remaining hemoglobins represent only a small percentage of the total hemoglobin. These hemoglobins are tentatively designated as embryonic hemoglobins based primarily on a comparison of their properties to those observed for hemoglobins from newly-hatched rainbow trout (Iuchi, I. (1973) Comp. Biochem. Physiol. 44B, 1087-1101). These two hemoglobins have isoelectric points near 5.9 and oxygen binding properties similar to those of the cathodal hemoglobins.

With the possible exception of one of the embryonic hemoglobins (for which globins were not obtained), all the hemoglobins are composed of two different types of globin chains. Six are ∝_2 β_2 tetramers, while two of the cathodal hemoglobins are hybrid tetramers of the type 〖∝∝'β〗_2 and ∝∝'ββ.

Red blood cells of cutthroat trout contain both ATP and GTP, suggesting that, in contrast to rainbow trout, both nucleotides may be important physiological modulators of hemoglobin oxygen affinity in this fish.

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