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 carrier exhibiting near physiological oxygen binding capability and devoid of nephrotoxic side effects, was previously found, by gel permeation, reversed-phase high performance liquid chromatography (RP-HPLC) and mass spectrometry, to consist of ca. 94% cross-linked product (reacted on the Lys 99 of two alpha-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 intermolecularly cross-linked dimers. However, conventional isoelectric focusing in carrier ampholyte buffers (CA-IEF) gave an unexpected spectrum of four major, almost equally represented, pI 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 other surface charge probes, such as immobilized pH gradients (IPG) and capillary zone electrophoresis (CZE) of native and denatured globin chains. IPGs of DCLHb gave the expected spectrum of bands, consisting of a main component (92%) with pl 7.337 and three additional minor bands, with lower pIs, representing 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 minor 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 alpha- and beta-globin chains in a 1:1 ratio in control Hb, resolved a free beta- and the alpha-alpha-dimer in DCLHb. In a 1:1 mixture of control and DCLHb, 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 species was ascertained by mass spectrometry.