Structural characterization of human hemoglobin crosslinked by bis(3,5-dibromosalicyl) fumarate using mass spectrometric techniques.
Diaspirin crosslinked hemoglobin (DCLHb) was analyzed by mass spectrometric-based techniques to identify the protein modifications effected by the crosslinking reaction with bis(3,5-dibromosalicyl) fumarate. DCLHb consists of two principal components. These components were isolated by size-exclusion chromatography and identified by measurement of their molecular weight using electrospray mass spectrometry and subsequent peptide mass mapping and mass spectrometric sequence analysis of their individual digests. Three major RP-HPLC fractions were observed from the major hemoglobin in DCLHb. Their MWs matched the MW of heme, intact hemoglobin beta-chain, and two hemoglobin alpha-chains crosslinked by a fumarate moiety, respectively. The minor HPLC peaks of DCLHb were also separated, and characterized by mass spectrometric methods. These minor components revealed additional details of the structural nature of covalent modification of DCLHb.
Yu
Z
Z
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0446, USA.
Friso
G
G
Miranda
J J
JJ
Patel
M J
MJ
Lo-Tseng
T
T
Moore
E G
EG
Burlingame
A L
AL
D000595:Amino Acid Sequence; D001241:Aspirin / Q000031:analogs & derivatives*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D006418:Heme / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D010449:Peptide Mapping
D003432:Cross-Linking Reagents; D006454:Hemoglobins; C021630:bis(3,5-dibromosalicyl)fumarate; D006418:Heme; D001241:Aspirin