ABSTRACT
The amino acid sequence of ß(I)-globin chain from Sindhi Krait (Bungarus sindanus sindanus) was determined to study the molecular evolution among snakes. The hemoglobin was isolated from the red blood cells and was analyzed by ion-exchange chromatography (IEX). The crude globin was subjected to reversed phased-high performance liquid chromatography (RP-HPLC) using C4 column. The N-terminal sequences of intact globin chains and tryptic peptides were determined by Edman degradation in a pulsed liquid gas phase sequencer using an online Phenylthiohydantoin analyzer. Sindhi Krait is expected to express three hemoglobin components that are composed of ß(II), ß(I), α(D) and α(A)-globin chains, as apparent by IEX, RP-HPLC and N-terminal sequence analyses. Sequence alignment and phylogenetic analyses of ß(I) globin chain from Sindhi Krait showed closest relationship with ß(I) globin chain from Rattlesnake, Water snake and Indigo snake. Interestingly, comparison of primary sequence of ß(I) globin chain of Sindhi Krait with human ß chain revealed 63 % similarity along with the retention of all heme contact points. Variations among the two sequences were prominent at αß contact points and in regions directly not important for function.
Subject(s)
Bungarus/genetics , Reptilian Proteins/chemistry , beta-Globins/chemistry , Amino Acid Sequence , Animals , Hemoglobins/chemistry , Hemoglobins/genetics , Hemoglobins/isolation & purification , Humans , Phylogeny , Reptilian Proteins/genetics , Reptilian Proteins/isolation & purification , Sequence Alignment , beta-Globins/genetics , beta-Globins/isolation & purificationABSTRACT
This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE) activity. The enzyme showed optimum activity at alkaline pH (8.5) and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5) inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5) inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km) for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.(AU)