The cross-linking of human Met-hemoglobin with [14C]dimethyl adipimidate

Abstract

Met-hemoglobin, cross-linked with [14C]dimethyladipimidate (cross-linking span 9 Å; 1 Å = 0.1 nm), yields four distinct molecular weight products (monomer, dimer, trimer, and tetramer) as observed on sodium dodecyl sulfate – polyacrylamide gels. The dimer species was purified to homogeneity by gel filtration. It was proteolytically degraded using a combination of Staphylococcus aureus V8 protease, pepsin, trypsin, and chymotrypsin. The resultant peptides were fractionated using gel filtration and ion-exchange chromatography methods. Two cross-links were unambiguously identified: (i) α1Lys7−α1Lys11 and (ii) β1Lys82−β2Lys82. The identified cross-links correlated well with the known structure of hemoglobin. However, attempts to isolate and identify a greater number of cross-linked peptides were unsuccessful owing to the complexity of the peptide mixtures. The complexity was a direct result of the chemical modification of the hemoglobin molecule. Therefore, attempts to employ chemical cross-linking as a means of examining sites of protomer contact within large oligomeric proteins should be approached with caution.