Purification, Crystallization and Preliminary analysis of Hemoglobin from Turkey (Meleagiris gallopova) and Domestic Pigeon (Columba livia)

Department of Crystallography and biophysics, University of Madras
â—‹Charles Packianathan Sundaresan Sigamani Neelakandan Kamariah Palani Kandavel Ponnuswamy Mondikalipudur Nanjapa goundar

Hemoglobin is an oxygen carrier protein in the red cells of the blood in mammals and other animals. The purification of the hemoglobins from both the sources were achieved by using the ion exchange column chromatography DEAE-Cellulose as the column material and the single peak of activity through SDS-PAGE electrophoresis. The hemoglobin crystals obtained by hanging drop vapor diffusion by using PEG 3350 as the precipitant in the reservoir. The data were collected using mar345image plate with the crystal to detector system of 100 mm at 293 K. The crystals of both the samples belong to orthorhombic space group P212121 and the cell dimensions are: a = 66.458 Å, b = 79.957 Å, c = 104.131 Å for turkey and a = 79.658 Å,b = 81.787 Å,c = 82.232 Å for pigeon. Evaluation of crystal packing parameter indicated that the lattice could accommodate one molecule per asymmetric unit (Matthews coefficient for turkey is 2.16(Vm(Å/Da)) and pigeon is 2.09 (Vm(Å/Da)) with the solvent contents are 43.13% and 41.26%). The structure solutions were obtained by molecular replacement method using Graylag goose hemoglobin (PDB id: 1FAW) as the starting model, using the program AmoRe implemented in CCP4 suite. The solution resulted in a conventional crystallographic R-factor of 33.0% and correlation coefficient of 77.5% for turkey and 35.1% and 75.7% for pigeon hemoglobin. The refinement of both the hemoglobins are underway using Refmac5.0.