Amino Acid Sequence Study on the Hemoglobin and Venom from Snakes Found in Pakistan

Abstract

Haemoglobin has been the extensively investigated protein, however few haemoglobin sequences from the class reptilian has been studied. The sequences reported in literature include that of Caiman (Caiman crocodylus) [1], the Nile crocodile (Crocodylus niloticus) [2] Mississippi crocodile (Alligator mississipiensis) [2], two turtles (Crysemys picta belli and Phrynops hilarii) [3], the lizards (Uromastix hardwicki and Varanus exanthematicus albigularis) [4, 5], Sphenodon (Sphenodon punctatus) [5] and an α chain from Viper (Vipera aspis) [6]. We have therefore investigated the primary structure of haemoglobin from cobra snake (Naja naja naja) to find out its relationship with other haemoglobin sequences. Experimental Isolation of haemoglobin: Cobra snake was dissected and the blood collected in heparinized tubes. Haemoglobin was isolated according to Drabkins method [7]. Blood was centrifuged at 3000 rpm for 15 min and the supernatant was discarded. Erythrocytes were washed thrice with physiological saline (0.9% NaCl) and lysed with distilled water at 4●C. the cell debris were removed by centrifugation and the clear supernatant containing haemoglobin was obtained. Preparation of Globin: Globin was prepared by acidic acetone precipitation [8]. 2% HCl acetone mixture was cooled to -18●C and the haemoglobin solution was added dropwise while constant stirring. The globin precipitated was separated by centrifugation and washed several times with cooled acetone. Globin was dissolved in water and lyophilized. Electrophoresis: Electrophoretic separation of the native globin chains achieved on polyacrylamide gel in presence of triton x-100 and 8M urea [9]. Separation of Globin Chains: The globin chains were separated on the column of CM-cellulose CM52 according to Clegg et al., [10]. A column (2.0x15cm) was packed and equilibrated with the starting buffer consisting of 8M urea, 0.05M sodium acetate, 0.2% 2- mercaptoethanol, pH 4.6. Globin was dissolved in starting buffer and applied on the column. Elution was performed with a linear salt gradient from 0-0.15M NaCl. The homogeneity of the separated chains was checked by urea gel electrophoresis in presence of triton x-100. Oxidation of Globin Chains: Purified globin chains were oxidized with performic acid by the method of Hirs et al., [11]. Globin chains were treated with cold performic acid prepared by mixing formic acid and hydrogen peroxide in 9:1 ratio. Reaction was stopped after 15 min by addition of distilled water. Acid was removed in vacuum and lyophilized. Cyanogen Bromide Cleavage: The chains were dissolved in 70% formic acid and cleavage was performed by addition of cyanogen bromide in a ratio of 40 mg/mg protein [12]. The reaction was carried out for 24 hrs at room temperature. The excess reagents were removed by evaporation in vacuum. The peptides were separated on a Sephadex G-50 column (1.5x140 cm) using 8M urea in 10% formic acid. Enzymatic digestion of globin chains: The oxidation chains were cleaved with TPCK-treated trypsin [13]. The oxidized chains were dissolved in distilled water and pH adjusted to 10.5 with ammonia. TPCK-trypsin was added in an enzyme to protein ratio of 5:100. Reaction was carried out for 2 hrs followed by digestion at pH9.5 for 1 hr. reaction was stopped by titrating to pH 4.0 with dilute acetic acid. The mixture was centrifuged. Separation of Tryptic Peptides: Separation of tryptic peptides was carried out by reserved phase-high performance liquid chromatography. A column of LiChrosorb RP-2 was equilibrated with 50 mM ammonium acetate buffer pH 6.0. Peptides were eluted with a linear gradient of acetonitrile 0-60% in 60 min at a flow rate of 1 ml/min. absorbance was monitored at 230 nm. Asp – Pro Cleavage: Globin chain was treated with 6M guanidine HCl and 70% formic acid. The reaction was carried out for 50 hrs at 42●C. the peptides were separated on a column of LiChrosorb RP-2. Elution was performed with 50mM ammonium acetate 10 % formic acid buffer and acetonitrile gradient. Amino Acid Analysis: The samples were hydrolysed with 6M HCl for 24 hrs at 110●C in evacuated tubes. These were dried in vacuum and dissolved in sodium citrate buffer pH 2.2. The amino acid composition was determined on a biotronik analyser LC-6001. Amino Acid Sequence Determination: N-terminal amino acid sequence of the native chains and of the peptides was determined by manual DABITC method [14, 15]. The native chains and the peptides were also subjected to automatic Edman degradation [16] in a gas phase sequencer Model 470A (applied Biosystem, calif, USA). The PTH derivatives of amino acids was analysed by HPLC using an online PTH analyser 120A (applied Biosystem).