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

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.