Barnett B. Rosenblum

Two-dimensional gel studies of genetic variation in the plasma proteins of Amerindians and Japanese

SummaryGenetic variation has been studied in plasma samples from 107 Amerindian children and their parents, and 110 Japanese children and their parents by means of two-dimensional polyacrylamide gel electrophoresis. Twenty-three polypeptides were scored; the identity of nine of these is at present still unknown. Genetic variation was encountered in 11 of these polypeptides. We have previously reported that the index of heterozygosity was 6.2±0.7% for 20 “randomly selected”, silver stained polypeptides scored for genetic variation in Caucasoids (Rosenblum et al. 1983b). For technical reasons only 11 of these 20 polypeptides could be routinely scored in preparations from the Amerindian samples. For these 11 polypeptides, the indices of heterozygosity in the three populations were: Amerindians, 4.5±0.6%; Japanese, 5.7±0.7%; Caucasoids, 8.0±1.1%. Even with these relatively small numbers some striking ethnic differences as regards individual polypeptides are apparent.

Identification and characterization of a human transthyretin variant.

An apparent Mr variant of plasma transthyretin (TTR), previously detected using 2-D PAGE, is the first reported occurrence of this type of human TTR variant. We characterized the variant TTR to determine the nature of this difference. Comparative tryptic peptide maps of variant and normal TTR and sequencing of peptides which differed indicated the variant contained a single amino acid substitution of valine for tyrosine at position 116. Because such a change requires two nucleotide substitutions, we postulate the variant arose through mutation in codon 116 of a heretofore unrecognized polymorphic or rare variant allele of TTR.

Separation of transferrin types in human plasma by anion-exchange high-performance liquid chromatography

An anion-exchange high-performance liquid chromatographic procedure was developed for the separation of transferrin in human plasma. The procedure allowed the four molecular forms of transferrin, which differ with respect to bound iron, to be separated from each other and from other plasma proteins. Transferrin variants, including B and D types, could also be identified using anion-exchange high-performance liquid chromatography. The approach followed for optimizing the separation of transferrin included identification of the peaks in the chromatogram by two-dimensional polyacrylamide gel electrophoresis. This approach could be extended to other proteins in plasma or biological fluids in order to optimize their separation.

BIOCHEMICAL APPROACHES TO MONITORING HUMAN POPULATIONS FOR GERMINAL MUTATION RATES: I. ELECTROPHORESIS*

The advent of relatively inexpensive and convenient techniques for identifying genetic variants of proteins on the basis of differences in molecular charge, thermostability, or, for enzymes, activity has opened a new chapter in the study of mutation. In this presentation, we shall speak to the progress being made in our understanding of human mutation rates and in monitoring programs because of the ability to readily detect charge and size variations in proteins; in a companion presentation, Dr. Mohrenweiser will address the progress being made with reference to the search for mutations affecting thermo stability or activity levels.

Hemoglobin Beirut (β126 Val ⏑ Ala [H4]) Synthesis in Erythroid Cultures

Abstract Hemoglobin Beirut is a β chain variant due to a neutral amino acid substitution. It is present in reduced amounts relative to βA in heterozygotes. Because HPLC has the potential for quantitating small amounts of hemoglobin, we examined the synthesis of Hb Beirut in erythroid colonies derived from the peripheral blood erythroid progenitor cells (BFUe) of an individual heterozygous for Hb Beirut and Hb A. The β globin chains (βBeirut and βA) were isolated and quantitated utilizing a combination of size exclusion, anion exchange and reverse phase HPLC. βBeirut accounted for 39.4% ± 4.6% of the total β chains in the accumulated hemoglobin and 43.3% ± 3.1% of the newly synthesized hemoglobin. The results indicate that Hb Beirut is synthesized in BFUe at a reduced rate which accounts for the reduced level of the variant hemoglobin in peripheral blood.