Interaction of erythroid spectrin with hemoglobin variants: implications in beta-thalassemia.
Among the few studies, producing contradictory results, done on the interaction of erythroid membrane skeletal spectrin with hemoglobin (Hb), none has been able to provide a quantitative estimate of the association of spectrin with Hb. In this work, studies on the interactions of erythroid spectrin with Hb have been elaborated upon using a novel fluorescence technique. The concentration-dependent change in the fluorescence intensity of fluorescein-conjugated spectrin (F-spectrin) in presence of oxy-Hb indicated binding with a dissociation constant of approximately 20 microM that has been directly evaluated from the increase in the extent of quenching of the fluorescein fluorescence of F-spectrin by reverse titration with the increasing concentrations of different Hb samples isolated from both normal and beta-thalassemic patients. The Hb compositions, with major components of the normal HbA, the fetal HbF, and the variant HbA2, of each individual were estimated using the Variant HPLC device of Bio-Rad. Results of the present study indicated that the dissociation constant, K(d), of spectrin binding to Hb decreased from 19.5 +/- 2 microM in normal individuals to of 6.5 +/- 0.5 microM in the presence of 73% HbA2 along with coeluted variants in the blood samples of patients suffering from beta-thalassemia, indicating differential interactions of the Hb variants with spectrin.
Biophysics Division, Saha Institute of Nuclear Physics, 37 Belgachia Road, Kolkata 700037, India.
Poppy
P
Datta
Sudipa Basu
SB
Chakrabarty
Amit
A
Chakrabarty
Abhijit
A
Chakrabarti
D016022:Case-Control Studies; D004912:Erythrocytes / Q000737:chemistry; D005319:Fetal Hemoglobin / Q000032:analysis / Q000378:metabolism; D006443:Hemoglobin A2 / Q000032:analysis / Q000378:metabolism; D006454:Hemoglobins / Q000032:analysis / Q000378:metabolism; D006455:Hemoglobins, Abnormal / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism*; D017186:Titrimetry; D017086:beta-Thalassemia / Q000097:blood*
D006454:Hemoglobins; D006455:Hemoglobins, Abnormal; D013049:Spectrin; D006443:Hemoglobin A2; D005319:Fetal Hemoglobin