Effects of methyl acetyl phosphate, a covalent antisickling agent, on the density profiles of sickle erythrocytes.
Methyl acetyl phosphate specifically acetylates valine-1, lysine-82, and lysine-144 in the 2,3-diphosphoglycerate binding cleft of hemoglobin S, thereby inhibiting its gelation (greater than 32 gm/dl) at pH 7.4. To extend these findings, the effect of methyl acetyl phosphate on the density of sickle cells has been evaluated by phthalate ester gradient centrifugation and by Larex-Percoll density centrifugation. After treatment with methyl acetyl phosphate (40% modification of the intracellular hemoglobin S), oxygenated sickle erythrocytes had a lowered density profile, as measured in a phthalate ester gradient. Thus 83% of untreated oxygenated sickle cells had densities greater than 1.098 gm/ml, whereas after treatment with methyl acetyl phosphate, 52% of the cells were in this density range. Under anaerobic conditions, methyl acetyl phosphate was even more effective in lowering cell density. For example, 50% of untreated deoxygenated cells had densities greater than 1.098 gm/ml, but none of the cells treated with methyl acetyl phosphate were this dense. For studies with Larex-Percoll density gradients, sickle erythrocytes were first separated into two fractions (densities greater than and less than 1.1 gm/ml) by Percoll-Hypaque centrifugation. The amount of oxygenated sickle cells exhibiting densities greater than 1.074 gm/ml decreased by about 32% on treatment with methyl acetyl phosphate. For deoxygenated sickle cells, treatment with methyl acetyl phosphate resulted in an average decrease of approximately 24% in the number of cells with densities greater than 1.074 gm/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Ueno
H
H
Rockefeller University, New York, NY 10021.
Yatco
E
E
Benjamin
L J
LJ
Manning
J M
JM
D000755:Anemia, Sickle Cell / Q000097:blood*; D000986:Antisickling Agents / Q000494:pharmacology*; D002458:Cell Fractionation; D002469:Cell Separation; D002499:Centrifugation, Density Gradient; D004906:Erythrocyte Count / Q000187:drug effects; D004913:Erythrocytes, Abnormal / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D006451:Hemoglobin, Sickle / Q000378:metabolism; D006801:Humans; D010100:Oxygen / Q000378:metabolism; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D014633:Valine / Q000378:metabolism
D000986:Antisickling Agents; D006451:Hemoglobin, Sickle; C014660:methylacetylphosphonate; D014633:Valine; D010746:Phosphonoacetic Acid; D010100:Oxygen