Abstract
Acetaldehyde reacted with hemoglobin at neutral pH and 37 degrees C to form adducts that were stable to dialysis and that were not reduced by sodium borohydride. Hemoglobin tetramers having 2, 3, and probably 4 molar eq of bound aldehyde were isolated by cation exchange chromatography. The sites of attachment of the aldehyde were the free amino groups of the N-terminal valine residues of the alpha and beta chains of hemoglobin. Derivatization of the beta chains caused a greater increase in the acidity of the hemoglobin than did derivatization of the alpha chains. Derivatization of the beta chains was also preferred over that of the alpha chains. Acetaldehyde derivatives of the N-terminal octapeptide of hemoglobin S (beta sT-1 peptide), Val-Gly-Gly, and tetraglycine were formed readily, contained 1 M eq of acetaldehyde/mol of peptide, and were not reduced by sodium borohydride. In contrast, Ala-Pro-Gly failed to form a 1:1 adduct with acetaldehyde. 13C NMR analysis of the peptide adducts formed with [1,2-13C]acetaldehyde indicated that tetrahedral diastereomeric derivatives were produced. The 13C chemical shifts of the adducts formed between hemoglobin and [1,2-13C]acetaldehyde were identical to those of the peptide adducts although resonances from the individual diastereomeric adducts at each hemoglobin site could not be resolved. The results cited above as well as other evidence indicate that acetaldehyde reacts with the amino termini of hemoglobin to form stable cyclic imidazolidinone derivatives. An exchange of acetaldehyde residues between peptides was also documented.
Highlights
Sites of attachment to hemoglobin of glucose [13], glyceraldehyde [14], and glycolaldehyde [15]
Selecting for further analysis the reaction readily dissociate by dilution or dialysis, and are susceptible mixture containing FMHin highest yield, preparative cation to and stabilized by borohydride reduction, our concern here exchange chromatography was performed by the method of is with aldehyde-modified proteins thatare formed more Abraham et al [20], giving the results shown in Fig. 1.Seven slowly than Schiff bases, are relatively stable to dialysis, and acetaldehyde-modified fractions were isolated, accounting for are notsusceptible to reduction by borohydrides
Having observed earlier that addition of the aldehyde to the p chains of hemoglobin & appeared to be limited to thefree amino group of the P-1 valine residue and that thiswas likely to be true of the addition of the aldehyde to a chains aswell, the appearance of a multiplicity of radioactive peaks suggested that, during the incubation with trypsin, acetaldehyde was transferred from the a-aminogroup of @T-1peptide, the N-terminal octapeptide of @ chains, to the a-amino group of N-terminal amino acid residues of the other peptides released by trypsin
Summary
Bins formed by reacting 1 mM hemoglobin A, with 5 mM [3H] In order to define the conditions necessary to generate acetaldehyde. The results, column 4, indicate that therelative acidity of acetaldehyde-modified hemoglobins, as evidenced by the order of their elution from cationic exchangers, increases with the number of chains to which the aldehyde is bound Having observed earlier that addition of the aldehyde to the p chains of hemoglobin & appeared to be limited to thefree amino group of the P-1 valine residue and that thiswas likely to be true of the addition of the aldehyde to a chains aswell, the appearance of a multiplicity of radioactive peaks suggested that, during the incubation with trypsin, acetaldehyde was transferred from the a-aminogroup of @T-1peptide, the N-terminal octapeptide of @ chains, to the a-amino group of N-terminal amino acid residues of the other peptides released by trypsin On this assumption the time of tryptic digestion was shortened in the expectation that less exchange would occur. With two sites of acetaldehyde addition to hemoglobin and two diastereomeric adducts forming a t each site, the intrinsically broad line widths of the resonances from the different protein bound adducts could not beresolved from one another even in thespectrum obtained after 40,000 accumulations
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