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KINETIC STUDIES ON THE OXIDATION OF MYOGLOBIN BY NITRITE. KINETIC STUDIES ON THE HEMOGLOBIN OF THE SEA LAMPREY, PETROMYZON MARINUS
Abstract
This thesis reports two separate kinetic studies on hemoproteins: the first, the oxidation of whale myoglobin, and the second, ligand binding and subunit aggregation kinetics of lamprey hemoglobin. The oxidation kinetics of sperm whale skeletal muscle oxymyoglobin by excess nitrite were studied. The oxidation curve showed an induction period prior to a rapid first order decay. The induction period varied inversely with the square of the nitrite concentration, whereas the rate of the first order decay was proportional to the nitrite concentration, but neither rate was affected by oxymyoglobin or metmyoglobin in the presence of excess nitrite. Various anions, such as cyanide, thiocyanate, azide and iodide, and some amino acids, such as tyrosine, tryptophan and cysteine, have inhibitory effects whereas other amino acids, such as histidine, methionine, threonine, and serine, have promotive effects on the rate of metmyoglobin formation. Intermediates were detected during the course of the oxidation. The extinction coefficients of one of the intermediates were estimated. The oxidation of deoxymyoglobin by excess nitrite showed a slower first order decay without the induction period and was not affected by dilute cyanide. The oxidation by nitrite of carboxymyoglobin, human oxyhemoglobin and its (beta)-chains, as well as Glycera monomeric oxyhemoglobin were studied for comparison with oxymyoglobin. The reaction of oxymyoglobin and nitrite is very complicated. The chain heterogeneity and subunit-subsunit interactions as well as dissociation of the tetramer cannot be the major causes of the sigmoidal nitrite oxidation kinetics. The kinetics of CO binding by the hemoglobin of the sea lamprey, Petromyzon marinus, have been followed in absorbance and light-scattering stopped-flow devices as well as by flash-photolysis. Lamprey hemoglobin is largely associated in the liganded form into monomers and is associated in the non-liganded form to dimers, and, at high concentration, tetramers. In order to describe the kinetics of ligand-binding, a kinetic scheme was proposed (M. E. Andersen and Q. H. Gibson, J. Biol. Chem. (1971) 246, 4790) which involved the 5 forms of the protein in ligand-binding and in protein association-dissociation reactions. These latter processes occur on the same time scale as the ligand-binding reactions. Rate constants for all reactions were obtained by Andersen and Gibson by fitting the extensive data obtained from the kinetic traces for absorbance changes. In the present study, these measurements were repeated, and good agreement with the absorbance changes were found. In addition, the light-scattering changes which occurred following a sudden drop in pH (pH 8.8 to 5.6) showed second-order kinetics with an association rate constant about one-tenth of that deduced by Andersen and Gibson from their curve-fitting operations. Other reactions associated with changes in protein aggregation show similar discrepancies and point to the utility of being able to make both light-scattering and absorbance change measurements in dissecting a complex ligand-binding mechanism.
Subject Area
Biochemistry
Recommended Citation
TAM, LEI-TING, "KINETIC STUDIES ON THE OXIDATION OF MYOGLOBIN BY NITRITE. KINETIC STUDIES ON THE HEMOGLOBIN OF THE SEA LAMPREY, PETROMYZON MARINUS" (1980). ETD collection for University of Nebraska-Lincoln. AAI8018678.
https://digitalcommons.unl.edu/dissertations/AAI8018678