Reaction of phenylhydrazine with erythrocytes. Cross-linking of spectrin by disulfide exchange with oxidized hemoglobin.
Phenylhydrazine causes deleterious oxidations of components of erythrocytes. These reactions and their effects on the mechanical properties of rabbit erythrocytes are investigated to provide insight into the mechanisms leading to destruction of oxidatively damaged erythrocytes. After 1 hr of incubation with phenylhydrazine, precipitated denatured protein (Heinz body protein) amounts to 25-30% of membrane protein, but deformability of erythrocytes as measured by filtrability is unchanged. After 4 hr of incubation filtrability drops sharply. Polymerization of spectrin and covalent binding of hemoglobin to spectrin, but no peroxidation of membrane lipids is observed. Precipitated protein amounts to 85-95% of membrane protein. It contains Fe, porphyrin and globin peptide in the proportion 1:1:1. Heinz body protein precipitated when hemoglobin is incubated under similar conditions has 90% of its sulfhydryl groups oxidized and no other amino acids than cysteine are destroyed. Addition of this Heinz body protein to erythrocyte ghosts causes polymerization of spectrin. Incubation of tetrathionate, a specific cross-linking agent, causes filtrability to drop sharply after about 80 min. This effect is similar to that observed after 4 hr incubation with phenylhydrazine, and is accompanied by polymerization of spectrin and band 3. The results indicate that cross-linking of membrane proteins by disulfide exchange with precipitated hemoglobin may play a major role in decreasing deformability during incubation with phenylhydrazine.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects*; D006366:Heinz Bodies / Q000378:metabolism; D006454:Hemoglobins / Q000378:metabolism*; D008054:Lipid Peroxides / Q000378:metabolism; D010084:Oxidation-Reduction; D010659:Phenylhydrazines / Q000633:toxicity*; D011817:Rabbits; D013049:Spectrin / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D004220:Disulfides; D006454:Hemoglobins; D008054:Lipid Peroxides; D010659:Phenylhydrazines; D013438:Sulfhydryl Compounds; D013049:Spectrin