Accession Number:

ADA223118

Title:

A Critical Examination of the Reaction of Pyridoxal 5-Phosphate with Human Hemoglobin Ao

Descriptive Note:

Corporate Author:

LETTERMAN ARMY INST OF RESEARCH PRESIDIO OF SAN FRANCISCO CA

Report Date:

1989-01-01

Pagination or Media Count:

13.0

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 phosphorus isotope of 30 or - 2 torr and a Hill coefficient of 2.3 or - 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. Excessive 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 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 or - 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. jg

Subject Categories:

  • Biochemistry
  • Organic Chemistry

Distribution Statement:

APPROVED FOR PUBLIC RELEASE