l-Amino acid oxidase from Cerastes vipera snake venom: Isolation, characterization and biological effects on bacteria and tumor cell lines

Walaa H Salama; Nihal M Ibrahim; Amr E El Hakim; Roqaya I Bassuiny; Manal M Mohamed; Fatma M Mousa; Mamdouh M Ali

doi:10.1016/j.toxicon.2018.06.064

l-Amino acid oxidase from Cerastes vipera snake venom: Isolation, characterization and biological effects on bacteria and tumor cell lines

Toxicon. 2018 Aug:150:270-279. doi: 10.1016/j.toxicon.2018.06.064. Epub 2018 Jun 10.

Authors

Walaa H Salama¹, Nihal M Ibrahim², Amr E El Hakim², Roqaya I Bassuiny², Manal M Mohamed², Fatma M Mousa³, Mamdouh M Ali³

Affiliations

¹ Molecular Biology Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, Egypt. Electronic address: walaahsalama82@gmail.com.
² Molecular Biology Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, Egypt.
³ Biochemistry Department, Genetic Engineering and Biotechnology Research Division, National Research Centre, Egypt.

PMID: 29898379
DOI: 10.1016/j.toxicon.2018.06.064

Abstract

A homodimeric l-amino acid oxidase enzyme (Cv-LAAOI) was isolated from the venom of Cerastes vipera (Egyptian Sand viper) using gel filtration followed by anion exchange chromatography. The molecular mass of Cv-LAAO is 120 kDa in its native form and 60 kDa in its monomeric form. The optimum enzyme activity was achieved on l-Leucine as a substrate in 50 mM buffer pH 7.5 at 50 °C. The Cv-LAAOI activity was significantly reduced by increasing the temperature over 40 °C, lost 75% of its activity at 60 °C and inhibited completely at 80 °C. The Cv-LAAOI attains the highest substrate specificity towards L-Met. The results have also indicated that Mn²⁺ enhances the enzyme activity by 10%, while Cu²⁺, Hg²⁺, Ni²⁺, Co²⁺ have suppressive effects on the Cv-LAAOI activity. On the other hand, EDTA has no significant effect on the enzyme activity. The kinetic parameters of Cv-LAAOI activity (K_m, K_cat and V_max) estimated on l-Leucine at pH 8 and 37 °C were found to be 2 mM, 12 S^-1 and 16.7 μmol/min/ml, respectively. In addition, the results have shown that Cv-LAAOI exhibits a significant bactericidal activity against gram-positive and gram-negative bacteria, particularly Staphylococcus aureus and Escherichia coli with MIC values of 20 μg/ml. Moreover, Cv-LAAOI has exhibited a considerable cytotoxic activity against breast cancer cell line (MCF-7) with IC₅₀ value 2.75 ± 0.38 μg/ml compared with different tumor cell lines (liver HepG2, lung A549, colon HCT116 and prostate PC3). Furthermore, Cv-LAAOI has triggered antiproliferative activity via extensive H₂O₂ generation as indicated by the increase in H₂O₂ and TBARS levels accompanied by the depletion in the catalase activity (CAT) in MCF-7 treated cells compared to the untreated ones. Thus, these findings clearly indicate that Cv-LAAOI has a selective cytotoxic effect on breast cancer cell line, demonstrating a great prospective for future use in cancer therapy.

Keywords: Antitumor; Bactericidal; Cerastes vipera viper; Cytotoxicity; Purification; Venom; l-amino acid oxidase.

MeSH terms

Animals
Anti-Bacterial Agents / chemistry
Anti-Bacterial Agents / pharmacology*
Antineoplastic Agents / chemistry
Antineoplastic Agents / pharmacology*
Bacteria / drug effects*
Cell Line, Tumor
Humans
Hydrogen-Ion Concentration
L-Amino Acid Oxidase / chemistry
L-Amino Acid Oxidase / metabolism*
Microbial Sensitivity Tests
Substrate Specificity
Temperature
Viper Venoms / chemistry
Viper Venoms / enzymology*
Viperidae / metabolism*

Substances

Anti-Bacterial Agents
Antineoplastic Agents
Viper Venoms
L-Amino Acid Oxidase