title: A critical examination of the reaction of pyridoxal 5-phosphate with human hemoglobin Ao.
abstract: 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 P50 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., greater than 10%). Excessive foaming and methemoglobin formation can be partially avoided using deoxyHbAo. Reduction with sodium cyanoborohydride is much gentler and gives solutions with less than 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 14C 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 P50 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 degrees C for one month, this preparation produces a mixture of HbAo and pyridoxylated HbAo with the original P50. 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 beta-chains. This is also the case for the cyanoborohydride reduced derivatives. When NaBH4 is used for the reduction, radioactively labeled PLP is found on both the alpha- and beta-chains.
authors.forename: M A
authors.initials: MA
authors.lastname: Marini
authors.forename: G L
authors.initials: GL
authors.lastname: Moore
authors.forename: R M
authors.initials: RM
authors.lastname: Fishman
authors.forename: R
authors.initials: R
authors.lastname: Jessee
authors.forename: F
authors.initials: F
authors.lastname: Medina
authors.forename: S M
authors.initials: SM
authors.lastname: Snell
authors.forename: A I
authors.initials: AI
authors.lastname: Zegna
mesh_terms: D006441:Hemoglobin A / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D010108:Oxyhemoglobins / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D010108:Oxyhemoglobins; D011732:Pyridoxal Phosphate; D006441:Hemoglobin A; C029774:oxyhemoglobin A