| Autor: |
Ogo, S, Focesi, A, Cashon, R, Bonaventura, J, Bonaventura, C |
| Zdroj: |
Journal of Biological Chemistry; July 1989, Vol. 264 Issue: 19 p11302-11306, 5p |
| Abstrakt: |
Spectrofluorometric techniques were used to quantify NADPH-hemoglobin interactions based on the quenching of NADPH fluorescence upon binding to hemoglobin. Fluorometric titrations were carried out with hemoglobin in varied states and with hemoglobins in which the β-chain anion site is altered. At pH 6.5 in 0.05 M4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, NADPH binds with high affinity, Kd= 1.03 µM, to deoxy human hemoglobin tetramers. Lower affinity binding of NADPH occurs as the β-chain anion-binding site is discharged by increasing the pH. Moreover, the cofactor binds in a 1:1 ratio to deoxy tetramers, inositol hexaphosphate binds competitively, and binding is decreased in hemoglobins whose structural alterations result in decreased effects of 2,3-diphosphoglycerate. The cofactor binds to oxidized (met) hemoglobin with an estimated Kdof 33.3 µMbut has little or no affinity for the oxy form. These results indicate that NADPH binds at the β-chain anion-binding site and can be considered as a fluorescent analog of 2,3-diphosphoglycerate. Fluorescence measurements gave no indication of NADPH binding to deoxygenated ferrous or ferric myoglobin. Reductive processes within the erythrocyte, such as reduction of met hemoglobin and hemoglobin-catalyzed enzymatic reactions, may be affected by the significant binding of the reduced cofactor to both deoxygenated and oxidized hemoglobin. Cofactor-hemoglobin interactions predict a shift in redox potential as red cells become oxygenated, which may account for unexplained oxygen-linked shifts in red cell metabolism. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
|
