R.Douglas Carter

A 13C-methylation study of glycophorin A in intact erythrocytes by 13C-NMR spectroscopy

N-terminal N alpha-[13C]monomethylamino derivatives for the N-terminal serine and leucine residues of glycophorins AM and AN, respectively, were obtained by reductively 13C-methylating homozygous human erythrocytes (MM, NN). The 13C-labeled glycophorins, AM and AN, were then isolated. A unique structural state was observed in solution reductively 13C-methylated glycophorin AM that was not observed in glycophorin AM derived from 13C-methylated erythrocytes. We attribute this state to the fact that some of the glycophorin AM forms a head-to-head dimer when subjected to reductive 13 C-methylation in aqueous solution. The 13C chemical shift data and pH titration data for the N-terminal [13C]dimethylamino and [13C]monomethylamino groups of glycophorin AM and AN derived from reductively 13C-methylated erythrocytes were in agreement with the chemical shift and titration data previously obtained for the N-terminal [13C]dimethylamino groups of solution reductively 13C-methylated glycophorins and related glycopeptides and peptides and N-terminal [13C]monomethylamino groups of related glycopeptides and peptides.

pH titration studies of 13C reductively methylated N-terminal serine of glycophorin AM

The 13C resonances of Nα,N-[13C]dimethylserine of partially 13C reductively methylated glycophorin AM were monitored as a function of pH at 45°C. For comparison, limited data are also presented for the pH dependence of the 13C resonances of Nα,N- [13C]dimethylserine of fully 13C reductively methylated deglycosylated glycophorin AM. The ‘major’ component of Nα,N- [13C]dimethylserine of glycophorin AM did not titrate, whereas the ‘minor’ component titrated with a pKa of 7.80 (Hill coefficient of 0.95). Similar results are also indicated for the Nα,N- [13C]dimethylserine resonances of 13C reductively methylated deglycosylated glycophorin AM.

13C n.m.r. study of the pH behaviour of N-methylated peptides related to the N-terminus of glycophorins

13C nuclear magnetic resonance spectroscopy (13C n.m.r.) was used to determine the pH titration parameters for the N-terminal Nα,N-[13C]dimethylamino and Nα,N-[13C]monomethylamino groups of glycophorins AM and AN, and some 28 related glycoproteins, glycopeptides and peptides. The results show that glycosylation of the Ser and Thr residues at positions 2, 3 and 4 of the glycophorins have a pronounced effect on the titration parameters. Substitution of amino acids 4 and 5 in the glycophorin sequence appears to minimally affect our titration parameters. Internal hydrogen-bonding involving the N-terminal Ser residue may explain some of the unusual pH titration results observed for glycophorin AM.

Determination of the mode of interaction of Mn2+ and Cu2+ with cis-inositol by 13C NMR spectroscopy

Abstract Natural abundance 13 C nuclear magnetic resonance spectroscopy ( 13 C NMR) was used to study the mode of binding of Mn 2+ and Cu 2+ to the cyclitol, cis -inositol. Resonance linewidths and the electron nuclear relaxation rates [( T 1 e ) −1 values] were used to establish that a unique binding site exists for these metal-ions on this cyclitol involving only the three axial hydroxyl groups. This work may aid in the development of new organometallic complexes used as paramagnetic relaxation agents in magnetic resonance imaging research.

Possible role of the carbohydrate residues on the structure of the n-terminus of glycophorin AM

Natural-abundance 13C nuclear magnetic resonance (13C-n.m.r.) was used to study the effect of monoglycosylation on the structure and dynamics of a pentapeptide related to the N-terminus of glycophorin AM. The results of this study indicate that a single point of glycosylation, on the pentapeptide, can significantly affect its structure. Moreover, glycosylation of this pentapeptide also affects its dynamic motion in solution. This study further defines the role that the carbohydrate residue plays in determining the structure about the N-terminus of glycophorin AM.

Structural studies of neuropeptides composed ofl-Asp andd-Glu by13C-NMR spectroscopy

Abstract13C-NMR spectral data are presented for four neuropeptides composed ofl-Asp, Ac-l-Asp, andd-Glu, which contain α and β peptide linkages. The data for the various compounds are compared to related dipeptides, one of which is a known neuropeptide, in order to gain structural information about these compounds. Electron-nuclear relaxation rates were used to elucidate the metal-ion binding sites of these species.

13C-NMR spectral study of reductively [13C]methylated glycophorin B

Glycophorin BN was reductively [13C]methylated and the 13C chemical shift of the N-terminal [13C]dimethyl-leucine residue was monitored as a function of pH. These results were compared to the pH-dependent chemical shift studies of the N-terminal [13C]dimethylleucine residues of intact glycophorin AN and N-terminal glyco-octapeptide AN. The results indicate that the titration data for [13C]dimethylleucine of glycophorin BN more closely resembles the titration data observed for the [13C]dimethylleucine residue of the N-terminal glyco-octapeptide AN rather than for the [13C]dimethylleucine residue of intact glycophorin AN. Integration of the 13C resonances indicated that glycophorin BN contains 3-4 lysine residues.

13C n.m.r. studies of the carbohydrate portion of glucoamylase from Aspergillus oryzae

Abstract Proton-decoupled, natural abundance 13C n.m.r. spectroscopy was used to investigate the carbohydrate structure and content of glucoamylase from Aspergillus oryzae. We found α- d -mannopyranose was the dominant sugar present (⋍91 residues). The Elson-Morgan assay showed that hexosamine was also present as a minor component (2.6% of the total carbohydrate). The intermannose linkages appear to be random. Integration data suggest that 41 α- d -mannopyranose residues are O-2 and O-3 glycosylated and 17 α- d -mannopyranose residues are involved in O-4 glycosylation. Treatment of glucoamylase with α-mannosidase appeared to remove all the carbohydrate residues present.

Structural elucidation of the reductive methylation by-product of glycophorin AM

Abstract The structural anomaly observed when glycophorin A M is subjected to reductive methylation with 13 C-enriched formaldehyde was investigated. A pyrylium salt was used to specifically block the ϵ-amino groups of the five lysine residues present in the glycophorin A M molecule. Consequently, methylation then only occurred at the α-amino group of the N-terminal Ser residue. Our spectral results from these studies strongly suggest that the by-product observed during the reductive methylation o glycophorin A is a head-to-head intermolecular dimer joined by the N-terminal Ser residues using the formaldehyde carbon as a methylene bridge.