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

Bossi, A; Patel, MJ; Webb, EJ; Baldwin, MA; Jacob, RJ; Burlingame, AL; Righetti, PG; (1999) Analysis of cross-linked human hemoglobin by conventional isoelectric focusing, immobilized pH gradients, capillary electrophoresis, and mass spectrometry. Electrophoresis , 20 (14) 2810 - 2817.

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Abstract

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 a

Type:Article
Title:Analysis of cross-linked human hemoglobin by conventional isoelectric focusing, immobilized pH gradients, capillary electrophoresis, and mass spectrometry
Additional information:UI - 20012302 LA - Eng RN - 0 (Cross-Linking Reagents) RN - 0 (Hemoglobins) PT - JOURNAL ARTICLE ID - RR01614/RR/NCRR DA -19991122 IS - 0173-0835 SB - M CY - GERMANY JC - ELE AA - Author EM - 200001
Keywords:analysis, pH, Biotechnology, chemistry, Chromatography, Cross-Linking Reagents, Electrophoresis, Capillary, Hemoglobins, Hydrogen-Ion Concentration, Isoelectric Focusing, Spectrum Analysis, Mass, Support, Non-U.S.Gov't, Support, U.S.Gov't, P.H.S., ANS
UCL classification:UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of)

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