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A critical examination of the reaction of pyridoxal 5‐phosphate with human hemoglobin Ao
Author(s) -
Marini Mario A.,
Moore Gerald L.,
Fishman Ronald M.,
Jessee Robert,
Medina Frank,
Snell Shawn M.,
Zegna Angelo I.
Publication year - 1989
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.360281204
Subject(s) - sodium cyanoborohydride , methemoglobin , chemistry , sodium borohydride , hemoglobin , sodium dithionite , chromatography , pyridoxal phosphate , pyridoxal , borohydride , phosphate , nuclear chemistry , inorganic chemistry , medicinal chemistry , organic chemistry , cofactor , enzyme , catalysis
Pyridoxylated normal adult human hemoglobin (HbAo) has been prepared using both oxygenated and deoxygenated HbAo at pH 6.8 and room temperature without the addition of Tris to produce a mixture with P 50 of 30 ± 2 torr and a Hill coefficient of 2.3 ± 0.1 similar to that of the isolated adult human hemoglobin from the red blood cell. Reduction of the pyridoxylated HbAo in the oxygen‐ligated form by sodium borohydride gives unacceptable levels of methemoglobin (i.e., > 10%). Exessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with < 5% methemoglobin. Both reducing agents give products with multiple components as shown by analytical chromatography. Radioautography on the isoelectric focusing gels of HbAo treated with 14 C pyridoxal 5‐phosphate (PLP) shows three major bands for the cyanoborohydride‐reduced derivatives and a much more complex mixture of labeled molecules after the sodium borohydride reduction. When pyridoxylated hemoglobin is prepared without reduction, the preparation, after passage through a mixed‐bed resin, contains 0.4 equivalents of PLP per heme, and has a P 50 of 30 ± 2 torr and an n value of 2.3 similar to the values found after reduction. Upon anion exchange resin chromatography, the PLP is removed, indicating that the reaction forms a reversible Schiff base. On standing at 4°C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P 50 . Methemoglobin increased to 3% during this incubation. After four months in the cold, the yield of a single chromatographic species is 70% with 20% methemoglobin. This fraction appears to be stable and can be passed through an anion exchange column without release of the PLP. Separation of the individual chains by reverse‐phase chromatography indicates that the addition of PLP to HbAo is directed solely to the β‐chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH 4 is used for the reduction, radioactively labeled PLP is found on both the α‐ and β‐chains.