Glycinamidation of aminoethylated CX, p, and y chains of adult and fetal human hemoglobins results in the attachment of a glycinamide residue to each free carboxyl group. The peptides which result from tryptic hydrolysis of such modified chains can be separated by column chromatography or filter-paper fingerprinting. Glycinamidation improves the yield of several tryptic peptides and is useful in determining the number and position of aspartyl and glutamyl residues in hemoglobins.
Koshland and co-workers (1-3) have demonstrated that a glycinamidyl residue can be attached to the free carboxyl groups in proteins by treating with glycinamide and a carbodiimide coupling reagent. Amino acid analysis following acid hydrolysis of such modified proteins will yield an extra glycine for each aspartyl and glutamyl residue present in the original protein plus an additional glycine residue for each free carboxyl terminus. This provides a method for differentiating the number of acidic amino acid residues from amide residues when compared with amino acid analyses of the unmodified protein.
This paper describes the application of the glycinamidation procedure to structural studies of hemoglobins.
The tryptic peptide patterns of glycinamidated alpha, beta, and gamma chains of normal human hemoglobins obtained by both filter-paper fingerprinting (4) and by automatic column chromatography (5) are illustrated. The modified peptides are identified and some of the advantages of glycinamidation of hemoglobin are discussed.
METHODS
Preparation of Globin Chains
Human hemoglobins from either normal adults or normal cord blood were purified by column chromatography on DEAE-Sephadex according