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Reexamination of the polymerization of pyridoxylated hemoglobin with glutaraldehyde
Author(s) -
Marini M. A.,
Moore G. L.,
Christensen S. M.,
Fishman R. M.,
Jessee R. G.,
Medina F.,
Snell S. M.,
Zegna A. I.
Publication year - 1990
Publication title -
biopolymers
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.556
H-Index - 125
eISSN - 1097-0282
pISSN - 0006-3525
DOI - 10.1002/bip.360290602
Subject(s) - chemistry , polymerization , tetramer , glutaraldehyde , chromatography , size exclusion chromatography , gel permeation chromatography , methemoglobin , polymer chemistry , hemoglobin , organic chemistry , polymer , enzyme
Pyridoxylated adult human hemoglobin (HbAo) was prepared using a one molar equivalent of pyridoxal 5‐phosphate (PLP) per heme and reduced with either NaCNBH 3 or NaBH 4 . A separate sample was pyridoxylated and passed through a mixed‐bed ion exchange column without reduction. All three preparations had a P 50 of 29 ± 2 torr and a cooperativity of n = 2.4 ± 0.1. These preparations, in both the oxy and deoxy forms, were then treated with 7 equivalents of glutaraldehyde per tetramer at pH 6.8 at 4°C and at room temperature. The polymerization invariably reduced the P 50 to 18 ± 2 torr with Hill coefficients of less than 2. These solutions, with or without further reduction using NaCNBH 3 , all retained the PLP in differing amounts (2–3 moles/tetramer). Methemoglobin concentrations were increased during the polymerization reaction. The normal pyridoxylation procedure, using sodium borohydride reduction, resulted in a number of different molecular species. Polymerization with glutaraldehyde caused a further proliferation of molecular species that could not be separated by anion exchange chromatography or by isoelectric focusing. The extent of polymerization, estimated by gel exclusion chromatography and SDS polyacrylamide gel electrophoresis, was from 40 to 50%. Analysis of the reverse phase chromatograms, which separate the heme and the α‐ and β‐chains, showed extensive polymerization and distribution of the radioactively labeled PLP on the protein for all preparations. All of the polymerized and pyridoxylated samples were unstable, and showed different chromatographic patterns after storage at 4°C for 1 month. Attempts to stabilize these preparations by further reduction with NaCNBH 3 gave products with a lower P 50 and lower cooperativity. When the reactions were conducted with a purified HbAo, heterogeneity was somewhat decreased compared to the normally used stroma‐free hemoglobin, but a large number of molecular species were still formed.