Total Fab Research Articles

The evolutionary sequence and quantities of different antigenic determinants of calf lens alpha crystallin

Immunochemieal cross-reactions between calf lens alpha crystallin and homologous proteins from different vertebrate classes were used for the study of antigenic determinants which, in the evolution of this protein, were derived from common ancestral forms. Determination of the sequence in which the different antigenic determinants evolved was based on estimation of the relative number of antigenic determinants shared by calf alpha crystallin with alpha crystallin from other vertebrate classes. For this estimation, samples of antiserum to calf lens alpha crystallin were absorbed with homologous lens proteins from each of the different vertebrate classes. The unabsorbed antiserum reacted with homologous proteins from all vertebrate classes. Each of the absorbed antisera showed a taxonomically different extent of residual cross-reaction. The species used for absorption were then ordered according to an increasing taxonomic extent of residual cross-reactions of the absorbed anti-calf antiserum, indicating a sequence of species with decreasing numbers of antigenic determinants shared with the mammalian protein. This sequence represents the increasing phylogenetic distances between the species used for absorption and the species used to produce the antiserum. This immunochemical determination showed that the phylogenetic sequence in which antigenic determinants present in the calf alpha crystallin molecule were formed, progressed from the primitive aquatic vertebrates agnatha, elasmobranchii and actinopterygii to the primitive land vertebrates and then to amphibia, reptilia, aves and mammalia. For the estimation of the relative amount (%) of determinants derived from different ancestral forms and transferred in evolution to calf alpha crystallin, 125I bound to a calf alpha crystallin immunoadsorbent after saturation with total 125I anti-calf alpha crystallin Fab fragments was compared to 125I bound to the same amounts of calf alpha crystallin after saturation with 125I Fab fragments of phylogenetically restricted specificity. Of all antigenic determinants in present-day calf alpha crystallin. approximately 42% were found also in the homologous lamprey protein. These determinants originated with the primitive agnatha which started the vertebrate subphylum 450 million years ago. Only about 15% originated with the mammalia, some 200 million years later, rellecting the slow evolutionary change of this protein molecule.

Separate Oxidase and Reductase Reaction Sites on Cytochrome c Demonstrated with Purified Site-specific Antibodies

Two of the four antibody populations elicited in rabbits by human cytochrome c were isolated in relatively pure form. One is the antibody population which is present in considerable excess over the other three; it was found to block the reaction of human cytochrome c with bovine cytochrome c oxidase, one combining site blocking the oxidation of 1 molecule of cytochrome c. The reaction of cytochrome c with the succinate-cytochrome c reductase is not blocked by this antibody. The other antibody population which has been purified is that controlled by the presence of isoleucyl residue 58 in human cytochrome c, which is replaced by a threonine in the otherwise identical Macaca mulatta protein. This antibody does not block the reactions with either the cytochrome c oxidase or the cytochrome c reductase. Mixtures of Fab fragments derived from all four antibody populations block both enzymic reactions. Thus, separate areas on the surface of cytochrome c are involved in its interaction with the oxidase and reductase segments of the respiratory chain. Antibodies or Fab fragments bound at sites other than those which completely prevent the enzymic reactions cause decreases in rate, probably due to the increase in the size of the reactant from a molecular weight of 12,500 for free cytochrome c to 5 to about 13 times that much for different cytochrome c-Fab fragment or antibody complexes. In addition, in the presence of low molar ratios of unfractionated total Fab fragments to cytochrome c, increases in the rate constant for the oxidase reaction were observed. These may be due to the blockage by an antibody population of an area on the cytochrome c surface which would otherwise bind to the oxidase in a nonreactive combination. No such rate increases were detected in the reductase reaction, which followed simple Michaelis-Menten kinetics, contrary to the complex situation with the oxidase reaction.