ELECTROSPRAY-IONIZATION MASS-SPECTROMETRIC STUDY OF THE MULTIPLE INTRACELLULAR MONOMERIC AND POLYMERIC HEMOGLOBINS OF GLYCERA-DIBRANCHIATA

The intracellular hemoglobin (Hb) of the marine polychaete Glycera dib ranchiata is comprised of two groups of globins differing in their pri mary structures and state of aggregation. About six electrophoreticall y and chromatographically distinct monomeric Hbs which have Leu as the distal residue, and an equal number of polymeric Hbs which have the u sual distal His, have been identified to date. Deconvolution of the el ectrospray ionization mass spectra (ESI-MS) of the Hbs and of their ca rbamidomethylated, reduced, and reduced/carbamidomethylated forms, usi ng a maximum entropy-based approach (MaxEnt), showed the presence of a t least 18 peaks attributable to monomer Hbs (14,500-15,200 Da) and an approximately equal number of polymer Hb peaks (15,500-16,400 Da). Al though the ratio of the monomer to polymer components in pooled Hb pre parations remained constant at 60:40, Hb from individuals had generall y less than 6 monomer and 6 polymer components;-2 of the 19 individual s appeared to be deficient in polymer Hbs. Taking into account possibl e fragmentations of the known monomeric and polymeric globin sequences , we estimate conservatively that there are 10 monomeric and an equal number of polymeric Hbs, the majority comprising a single free Cys. Su rprisingly, the calculated mass of the sequence deduced from the high- resolution monomer Hb crystal structures does not correspond to any of the observed masses. ESI-MS of the monomer Hb crystal revealed 11 com ponents, of which 5, accounting for 67% of total, were related to the three major sequences GMG2-4. These findings underline the need for ro utine mass spectrometric characterization of all protein preparations. The complete resolution of the Glycera Hb ESI-MS using MaxEnt process ing illustrates the power of this method to resolve complex protein mi xtures.