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Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

Jagadeesan G, Malathy P, Gunasekaran K, Harikrishna Etti S, Aravindhan S - Acta Crystallogr F Struct Biol Commun (2014)

Bottom Line: Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates.In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins.Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Presidency College, Chennai 600 005, India.

ABSTRACT
Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

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10% native PAGE gel stained with Coomassie Blue. Lane 1, cormorant haemolysate Hb.
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fig1: 10% native PAGE gel stained with Coomassie Blue. Lane 1, cormorant haemolysate Hb.

Mentions: Fresh whole blood from great cormorant was collected, transferred immediately to 0.01% EDTA to avoid clotting and stored at 4°C. Red blood cells (RBC) were isolated from blood by centrifugation at 1398g for 20 min at 4°C (Neelagandan et al., 2007 ▶). Isolated RBC were washed thrice with two volumes of 0.9%(w/v) saline solution and haemolyzed by the addition of three volumes of ice-cold Millipore water. Subsequent centrifugation at 5590g for 1 h yielded cell-free haemoglobin solution as the supernatant. The isolated protein was extensively dialyzed against distilled water for 24 h to remove trace salts and the sample was then loaded onto a DEAE-cellulose anion-exchange chromatography column (15 × 1.5 cm) equilibrated with 50 mM sodium phosphate buffer pH 7. The column was eluted with the same buffer, followed by stepwise elution with various concentrations of sodium chloride (NaCl) solution. A single peak obtained at 0.1 M NaCl was collected at a rate of 2 ml min−1. A small portion of the sample was used to check for protein content using Bradford assay (Bradford, 1976 ▶) and the purity was assessed by native gel electrophoresis (Laemmli, 1970 ▶; Fig. 1 ▶).


Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of great cormorant (Phalacrocorax carbo) haemoglobin.

Jagadeesan G, Malathy P, Gunasekaran K, Harikrishna Etti S, Aravindhan S - Acta Crystallogr F Struct Biol Commun (2014)

10% native PAGE gel stained with Coomassie Blue. Lane 1, cormorant haemolysate Hb.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231857&req=5

fig1: 10% native PAGE gel stained with Coomassie Blue. Lane 1, cormorant haemolysate Hb.
Mentions: Fresh whole blood from great cormorant was collected, transferred immediately to 0.01% EDTA to avoid clotting and stored at 4°C. Red blood cells (RBC) were isolated from blood by centrifugation at 1398g for 20 min at 4°C (Neelagandan et al., 2007 ▶). Isolated RBC were washed thrice with two volumes of 0.9%(w/v) saline solution and haemolyzed by the addition of three volumes of ice-cold Millipore water. Subsequent centrifugation at 5590g for 1 h yielded cell-free haemoglobin solution as the supernatant. The isolated protein was extensively dialyzed against distilled water for 24 h to remove trace salts and the sample was then loaded onto a DEAE-cellulose anion-exchange chromatography column (15 × 1.5 cm) equilibrated with 50 mM sodium phosphate buffer pH 7. The column was eluted with the same buffer, followed by stepwise elution with various concentrations of sodium chloride (NaCl) solution. A single peak obtained at 0.1 M NaCl was collected at a rate of 2 ml min−1. A small portion of the sample was used to check for protein content using Bradford assay (Bradford, 1976 ▶) and the purity was assessed by native gel electrophoresis (Laemmli, 1970 ▶; Fig. 1 ▶).

Bottom Line: Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates.In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins.Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Presidency College, Chennai 600 005, India.

ABSTRACT
Haemoglobin is the iron-containing oxygen-transport metalloprotein that is present in the red blood cells of all vertebrates. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of avian haemoglobins. Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on cormorant (Phalacrocorax carbo) haemoglobin. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 3350, NaCl and glycerol as precipitants. The crystals belonged to the trigonal system P3₁21, with unit-cell parameters a=b=55.64, c=153.38 Å, β=120.00°; a complete data set was collected to a resolution of 3.5 Å. Matthews coefficient analysis indicated that the crystals contained a half-tetramer in the asymmetric unit.

Show MeSH
Related in: MedlinePlus