Ueno H, Pospischil M A, Kluger R, Manning J M
J Chromatogr. 1986 May 30;359:193-201. doi: 10.1016/0021-9673(86)80073-5.
A novel acetylating agent, methyl acetyl phosphate (MAP), has been designed to react with a nucleophile near an anion binding site of proteins. We examined the effect of MAP on hemoglobin (Hb), which has a well defined binding site for 2,3-diphosphoglycerate (DPG), to determine whether this reagent recognizes the DPG binding site. The progress of the reaction was monitored by ion-exchange high-performance liquid chromatography (HPLC) on a TSK CM-SW column. Modified Hb was initially chromatographed on CM-52 and then separated into its component chains. The alpha- and beta-chains from modified and unmodified Hb were digested by TPCK-trypsin. The peptide mixtures were chromatographed on Whatman ODS-3 reversed-phase HPLC columns and the peptide maps of modified and unmodified chains were compared. The peaks formed by the modification with MAP were further purified on YMC ODS-S5 columns and then subjected to amino acid analysis on a Dionex D-500 instrument after acid hydrolysis. We found that the newly formed peptides are beta T1 and beta T14 + 15 and that the loss of a peptide corresponding to beta T9 and beta T 10 + 11 is significant. No change in the alpha-chains was observed. The results suggest that MAP is indeed specific for the DPG binding site, as the above peptides contain the amino acid residues involved in the binding of DPG. We have assigned the acetylation sites as Val-1(beta), Lys-82(beta) and Lys-144(beta).
