Isolation and Characterization of Angiotensin-Converting Enzyme Inhibitors from Agkistrodon halys halys Venom
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ISOLATION AND CHARACTERIZATION OF ANGIOTENSIN-CONVERTING ENZYME INHIBITORS FROM Agkistrodon halys halys VENOM
V. M. Lcvov,† E. S. Sadykov, E. S. Yunusova, and A. V. Shkinev*
UDC 547.993
The peptide fraction of venom from the Eastern pit viper Agkistrodon halys halys (Crotalidae) that was obtained by gel-filtration over Sephacryl S-100 HR exhibited high activity as an angiotensin-converting enzyme (ACE, EC 3.4.15.1) inhibitor. Separation of the materials over TSK HW-40F and SP-Sephadex C-25 produced purified (chromatographically pure) ACE inhibitors (ACEI) of molecular weight up to 2.5 kDa and IC50 values in the range 0.48–15.3 Pg/mL. Keywords: pit-viper venom, fractionation, angiotensin-converting enzyme inhibitors, peptides, inhibition analysis. Angiotensin-converting enzyme (ACE) inhibitors (ACEIs) are the leading drugs for treating cardiovascular diseases. Structural data of low-molecular-weight peptides from snake venoms [1] modified by sulfhydryl, carboxyl, and phosphate groups (captopril, enalapril, and fosinopril, respectively) were used to create them [2]. These drugs are not very selective for ACE [1, 2] so that their systemic administration produces uncontrolled side effects (cough, skin rashes, vessel edema, etc.). Hence, the search for and comparisons of new ACEIs with minimal effects on other in vivo systems from various sources including snake venoms is timely [1, 3–9]. The activity of ACEIs from venoms was shown earlier [6] to decrease in the series pit-viper > common northern viper > blunt-nosed viper > steppe viper. Therefore, research on their isolation was performed using pit-viper venom. The ACEI fraction was obtained using chromatography of the venom on a high-resolution calibrated column of Sephacryl S-100 HR 16/60, separating them as a sum of peptides of different molecular weights (MWs) (Fig. 1). According to gel-filtration, a fraction (Aghh-III) of peptides with MWs 8.0–0.5 kDa and 6.6, 4.5, and 1.1 kDa was formed. We found earlier during screening of ACEIs in venoms of Central Asian snakes [5, 6] that this fraction contained lowmolecular-weight peptides that were characterized by weak absorption at 280 nm that was apparently due to an insignificant content of aromatic amino acids. They did not appear as separate peaks on densitograms. However, their weight content was substantial. Electrophoresis of the studied fraction (under denaturing conditions) did not detect in it discrete components with MWs > 2.0 kDa. This suggested that the components with MW 8.0–2.0 kDa that were observed by gel-filtration were aggregates of peptides of lesser MW. Data on the hydrolysis of Aghh-III by HCl (5.7 N) or a mixture of proteolytic enzymes (trypsin, chymotrypsin, carboxypeptidase Y) argued in favor of the peptide nature of the identified inhibitors. As a result, the ACEI activity decreased by an average of 80%. The remaining 20% of the activity was associated with the presence in the studied materials of an N-terminal pyroglutamate residue that was not hydrolyzed under the experimental conditions and wa
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