Detection of neutral amino acid substitutions in proteins.
The field of biochemical genetics relies heavily upon the detection by electrophoresis of genetically determined variants of proteins. Most of these variants differ by substitutions that involve charged amino acids. Genetic variants of another large class, ones that involve substitutions among neutral amino acids, are not easily detected and are often ignored. Ampholyte isoelectric focusing in some cases can separate proteins indistinguishable by standard electrophoresis, including genetic variants of mouse hemoglobins that differ only by neutral amino acid substitutions. A revolutionary variation of isoelectric focusing, in which gradients covering a small pH range are fixed into place in a polyacrylamide gel, provides greater resolution of these nearly identical proteins. Mouse hemoglobin tetramers that differ only by the substitution of alanine for glycine in the alpha-globin chains are resolved by several millimeters with the new technique; by comparison, these tetramers are imperfectly resolved on a standard pH 7-9 isoelectric focusing gel. This improved technique of isoelectric focusing was used to identify a variety of previously unreported genetic variants of mouse hemoglobin alpha chains. Immobilized gradients tailored to the requirements of the proteins being analyzed will extend greatly the ranges of protein variations that can be easily recognized for diverse applications, including genetic quality-control analyses and in studies of genetics, mutagenesis, and evolution.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D004586:Electrophoresis; D005914:Globins / Q000032:analysis / Q000235:genetics; D006454:Hemoglobins / Q000032:analysis*; D007525:Isoelectric Focusing; D051379:Mice; D008815:Mice, Inbred Strains; D009154:Mutation
D000596:Amino Acids; D006454:Hemoglobins; D005914:Globins