Biennial Report 2005-2007 - Saha Institute of Nuclear Physics

202 Biennial Report 2005-07that of the β-globin in the hemoglobin disorder, β-thalassemia.Poppy Datta, Sudipa Chakraborty†, Amit Chakraborty†, Abhijit ChakrabartiSG6.1.1.12 Membrane interactions of hemoglobin variants, HbA, HbE, HbF and globinsubunits of HbA : effects of aminophospholipids and cholesterolThe interaction of hemoglobin with phospholipid bilayer vesicles (liposomes) has been analyzed inseveral studies to better understand membrane-protein interactions. However, not much is knownon hemoglobin interactions with the aminophospholipids, predominantly localized in the innerleaflet of erythrocytes, e.g. phosphatidylserine (PS), phosphatidylethanolamine (PE) in membranescontaining phosphatidylcholine (PC). Effects of cholesterol, largely abundant in erythrocytes, havealso not been studied in great details in earlier studies. This work describes the study of the interactionsof different hemoglobin variants HbA, HbE and HbF and the globin subunits of HbAwith the two aminophospholipids in the presence and absence of cholesterol using absorption measurementson the autoxidation of the hemoglobin variants and leakage of membrane entrappedcarboxyfluorescein (CF), induced by them. Results indicate preferential oxidative interaction ofHbE and alpha-globin subunit with unilamellar vesicles containing PE and PS compared to normalHbA. Cholesterol was found to stabilize such oxidative interactions in membranes containing boththe aminophospholipids. HbE and alpha-globin subunits were also found to induce greater leakageof membrane entrapped CF. HbE was found to induce fusion of membrane vesicles containingcholesterol and PE when observed under electron microscope. Taken together, these findings mightbe helpful in understanding the oxidative stress related mechanism(s) involved in the prematuredestruction of erythrocytes in peripheral blood, implicated in the hemoglobin disorder, HbE /betathalassemia.Poppy Datta, Sudipa Chakraborty†, Amit Chakraborty†, Abhijit ChakrabartiSG6.1.1.13 Loss of phospholipid membrane asymmetry and sialylated glycoconjugatesfrom erythrocyte surface in HbEβ-thalassemia : implications in eryptosisPhosphatidyl serine (PS) is mostly localized in the inner leaflet of the normal RBC membrane.However, it has been shown by our group and others that the PS exposed subpopulation is markedlyhigh in HbEβ-thalassemia and other forms of thalassemias. Older populations of RBCs have beenshown to exhibit more PS in the outer leaflet than the younger counterparts. On the other hand,sialic acid residues residing on the RBC surface have been shown to gradually reduce during ageingof erythrocytes in the peripheral blood. We have therefore studied the PS asymmetry along withsialic acid levels of the normal and different thalassemic RBCs. Young and old erythrocytes wereseparated depending on their densities by self-forming Percoll gradient. FITC labeled Annexin Vwas used as a marker for PS and FITC labeled WGA as a marker for sialic acid in flow cytometry.The PS exposed population in older RBC was observed to be higher compared to younger onein case of normal as well as in β-thalassemia and homozygous E disease. However, the reversetrend was observed in HbEβ-thalassemia. The reduction of sialic acid residues on the cell surfaceglycoconjugates upon ageing was more drastic in HbEβ-thalassemia, compared to that in normal.This reduction of sialic acid levels could be due to the vesiculation of cell surface membranes