The stepwise mutation model: an experimental evaluation utilizing hemoglobin variants.

The stepwise mutation model of Ohta and Kimura (1973) was proposed to explain patterns of genetic variability revealed by means of electrophoresis. The assumption that electrophoretic mobility was principally determined by unit changes in net molecular charge has been criticized by Johnson (1974, 1977). This assumption has been tested directly using hemoglobin. Twenty-seven human hemoglobin variants with known amino acid substitutions, and 26 nonhuman hemoglobins with known sequences were studied by starch gel electrophoresis. Of these hemoglobins, 60 to 70% had electrophoretic mobilities that could be predicted solely on the basis of net charge calculated from the amino acid composition alone, ignoring tertiary structure. Only four hemoglobins showed a mobility that was clearly different from an expected mobility calculated using only the net charge of the molecule. For the remaining 30% of hemoglobins studied, mobility was determined by a combination of net charge and other unidentified components, probably reflecting changes in ionization of some amino acid residues as a result of small alterations in tertiary structure due to the amino acid substitution in the variant. For the nonhuman hemoglobins, the deviation of a sample from its expected mobility increased with increasing amino acid divergence from human hemoglobin A.-It is concluded that the net electrostatic charge of a molecule is the principal determinant of electrophoretic mobility under the conditions studied. However, because of the significant deviation from strict stepwise mobility detected for 30 to 40% of the variants studied, it is further concluded that the infinite-allele model of Kimura and Crow (1964) or a "mixed model" such as that proposed by Li (1976) may be more appropriate than the stepwise mutation model for the analysis of much of the available electrophoretic data from natural populations.