STRUCTURE DETERMINATION OF CORMORANT (PHALACROCORAX CARBO)
HAEMOGLOBIN THROUGH X-RAY CRYSTALLOGRAPHY
The Great Cormorant is called large cormorant in India and has an extremely large distribution across the world. This chapter describes about the 3D-structure determination of Cormorant hemoglobin (Hb) using X-ray crystallographic method. Avian often show efficient oxygen management to meet the demands of their metabolism. However, structural studies of avian Hb are rather scanty to understand the mechanism involved in meeting the high oxygen demand.
Red blood cells collected from cormorant were separated by centrifugation, followed by cell lysis with ice-cold deionized water to obtain haemoglobin. Further purification was carried out by Ion- exchange chromatography using DEAE-cellulose as the matrix material. The retained Hb is eluted using Sodium Phosphate buffer (pH 7.0) containing 100 mM Nacl. The purity of the protein was evaluated by SDS and Native PAGE. Crystallization by hanging drop vapor diffusion method yielded good quality crystals at 289 K. Diffraction data were collected at Rigaku RU-
H3R rotating anode generator of Mar345 image plate detector system using CuKα radiation
(1.5418Å) operated at 40 kV and 50 mA. Intensity measurements were processed and analyzed using AUTOMAR programme package and scaled using SCALEPACK . The crystals belong to trigonal system P3
1
21 with cell dimension a = b= 55.64 Å and c = 153.38 Å and
β
=120.00°.
The structure was solved by molecular replacement method using grey lag goose Hb structure as the template. The rotational and translational searches were calculated using AMORE .
Refinements were carried out in REFMAC program, where initial few rounds of refinements result in lowering the R factor
and R free
, which reduces further after adding water molecules to 0.19 and 0.26, respectively. The coordinate were deposited in RCSB-PDB with accession code 3WR1.