Analysis of cross-linked human hemoglobin by conventional isoelectric focusing, immobilized pH gradients, capillary electrophoresis, and mass spectrometry

Diaspirin cross-linked hemoglobin (DCLHb), a hemoglobin-based oxygen carrie r exhibiting near physiological oxygen binding capability and devoid of nep hrotoxic side effects, was previously found, by gel permeation, reversed-ph ase high performance liquid chromatography (RP-HPLC) and mass spectrometry, to consist of ca. 94% cross-linked product (reacted on the Lys 99 of two a lpha-chains), accompanied by ca. 6% cross-linked Hb, which also reacted on the Lys 132 and/or Lys-144 of the beta-chains and a small amount of intermo lecularly cross-linked dimers. However, conventional isoelectric focusing i n carrier ampholyte buffers (CA-IEF) gave an unexpected spectrum of four ma jor, almost equally represented, p/species in the pH range of 6.82-7.01, a band of mid-intensity with a pi of 7.11, and two minor components with pls of 6.73 and 6.77. This extraordinary polydispersity was reevaluated by othe r surface charge probes, such as immobilized pH gradients (IPG) and capilla ry zone electrophoresis (CZE) of native and denatured globin chains. IPGs o f DCLHb gave the expected spectrum of bands, consisting of a main component (92%) with p/7.337 and three additional minor bands, with lower p/s, repre senting ca. 8% of the total. These data were in agreement with CZE profiles of native DCLHb, which resolved, in addition to the main DCLHb peak, 3-4 m inor components representing ca. 10% of the total. Also, CZE of denatured, heme-free globin chains gave the expected pattern with only traces of minor , extrareacted species. The latter technique, in addition to resolving alph a- and beta-globin chains in a 1:1 ratio in control Hb, resolved a free bet a- and the alpha-alpha-dimer in DCLHb. In a 1:1 mixture of control and DCLH b, three peaks were observed, eluting in the order alpha-, alpha-alpha- and beta-globin chains. The identity of the major DCLHb and of the minor speci es was ascertained by mass spectrometry.