High-pressure liquid chromatography and field desorption mass spectrometry of heme a, Heme a dimethyl ester and acetyl heme a dimethyl ester

Abstract

Field desorption mass spectroscopy was shown to be a valid technique for determining the molecular weights of hemins and hemin esters, as well as of porphyrins. The observed base peaks of ligand-free protoheme IX, protoheme IX dimethyl ester, and protoporphyrin IX dimethyl ester correspond well to the molecular weights of these compounds and the base peak for hematoporphyrin corresponds to the molecular weight of this porphyrin minus two molecules of water. The technique was employed to confirm the molecular weights of heme a, heme a dimethyl ester, and acetyl heme a dimethyl ester. Heme a dimethyl ester was prepared by reaction of heme a with trimethyloxonuim tetrafluoroborate and purified by high-pressure liquid chromatography. The isolated product was converted to the acetylated derivative by reaction with acetic anhydride and was subsequently purified by high-pressure liquid chromatography. A field desorption spectrum of heme a shows a base peak at 582 which is in agreement with previous deductions of the structure of this prostetic group. Base peaks of the heme a ester and its acetylated derivative demonstrate that the two carboxyl groups have been methylated and the single hydroxyl group has been acetylated without alteration of the molecule.