The hemoglobin variant South Florida has been shown by protein sequencing and fast-atom-bombardment mass spectroscopy to have a substitution ofmethionine for the NH2-terminal valine of the (8-globin chain. In addition, there was complete retention of the initiator methionine on the mutant polypeptide. Approximately 20% of the protein was acetylated at the NH2 terminus of the (3 chain. A search of a comprehensive data bank of protein and gene sequences revealed 84 unrelated vertebrate proteins that have not undergone cleavage of leader sequences. A highly nonrandom distribution of residues at the NH2 termini of these proteins predicts removal of the initiator methionine as well as NH2-terminal acetylation. Proteins that undergo removal commonly have serine, alanine, glycine, or valine, as the NH2-terminal residues. The first three residues favor N-acetylation. Proteins that retain the initiator methionine commonly have a charged residue or methionine at the second position. Information on Hb South Florida and other hemoglobins coupled with this survey of primary sequence provides insights into the NH2-terminal processing of proteins.The biological properties of a large number of proteins are significantly altered by cotranslational and posttranslational modifications. In eukaryotes, the initiation of translation takes place at an AUG codon on mRNA (1). The NH2-terminal methionine residue is generally cleaved from the nascent polypeptide by an aminopeptidase (2). Soon thereafter the new NH2-terminal residue is often acetylated at the a-amino group (3,4). Only fragmentary information on NH2-terminal processing is available and, therefore, the molecular mechanism is still poorly understood. It has been suggested that cleavage of the initiator methionine is prevented when the adjacent residue is a charged amino acid (5). The MATERIALS AND METHODSPhosphate-free hemolysates (6) were prepared from heparinized blood samples. Hemolysate (1-2 g of hemoglobin) was analyzed on a column (5 x 22 cm) of Bio-Rex 70 (Bio-Rad) cation-exchange resin as described (7). The "Hb Al," fraction was dialyzed vs. 0.25 M ammonium acetate, pH 8.5, and rechromatographed on a column (2 x 6 cm) of Glycogel B boronate affinity resin (Pierce) (8). The non-retained Hb fraction from the affinity column and Hb A0 fraction from Bio-Rex chromatography were converted to globin by acid/acetone precipitation (9) and separated into a and /3 subunits by carboxymethyl-cellulose chromatography in 8 M urea. After aminoethylation (10), the isolated chains were digested with trypsin (11 Tryptic peptides were dissolved in 0.5% acetic acid/glycerol, 1:1 (vol/vol) solution. About 2 ,ug of the sample was applied per analysis.Abbreviations: FAB-MS, fast-atom-bombardment mass spectroscopy; RP-HPLC, reverse-phase HPLC. 8448The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
An 8.75-yr-old Caucasian boy was discovered to have a markedly elevated (14.8%) hemoglobin A1c (HbA1c) as estimated by ion-exchange chromatography (Bio Rex 70). Glycohemoglobin (GHb) measured by a colorimetric method with thiobarbituric acid (TBA) was normal (equivalent to a 6.4% HbA1c). Nondiabetic quantities of GHb were found with affinity chromatography, and the glucose tolerance test was normal. Intensive efforts to identify an abnormal variant hemoglobin by several electrophoretic methods were unsuccessful. A family survey identified a similar abnormality in 11 other individuals, revealing an autosomal-dominant pattern. None of the affected individuals had any other hematologic abnormality. Structural analysis in one family member revealed a new hemoglobin variant (approximately 45% of the total hemoglobin) with the substitution of methionine for valine at the beta-NH2-terminal. In addition, the initiator methionine residue was preserved. Approximately 20% of the variant hemoglobin was modified by acetylation of the NH2-terminal methionine. The modified variant coeluted with HbA1c. We suggest that patients who do not have an explanation for their elevated HbA1c should have GHb measured by the TBA method or affinity chromatography because hemoglobin electrophoresis does not identify this confounding artifact.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.