Ionizing radiation target groups of band 3 inserted into egg lecithin liposomes as determined by Raman spectroscopy.

Abstract

The purified integral membrane protein, band 3, from human erythrocytes was inserted into egg lecithin liposomes. The insertion of band 3 was determined from thermal transition data from the analysis of the C--H stretching region bands recorded at temperatures from 25 to -22 degrees C. Raman spectra show that band 3 considerably broadens and lowers the thermal transition of egg lecithin liposomes, suggesting the insertion of band 3. The band 3-inserted liposomes were irradiated with gamma-rays (40 Gy) and the radiation target groups were determined by the analysis of the structural sensitive Raman bands in the 1600-1700 cm-1 (amide I), 1200-1300 cm-1 (amide III) and 550-1030 cm-1 (side chain amino groups) regions. The radiation-sensitive groups as identified from Raman spectra in the region 550-1030 cm-1 are tyrosines and cysteines. The radiation-induced changes in the secondary structure were determined from amide I and III bands. Quantitative estimation using the curve fitting method shows that band 3 contains 44% total helix, 48% beta strand and 8% undefined plus turns (error +/- 4%). The secondary structure changes to 35% total helix, 42% total beta-strand and 23% turned and undefined upon irradiating band 3 containing liposomes. We suggest that ionizing radiation preferably damages tyrosine and cysteine side chain residues and reduces the amount of alpha-helical configuration of band 3.