(Original Article) Assessment of L-Amino Acid Oxidase Activity in Snake Venoms by Prussian Blue Agar Assay: Purification and Characterization of the King Cobra Enzyme

Wichit Thaveekarn; Jureeporn  Noiphrom; Orawan  Khow; Sirisak  Sathorn

Authors

Wichit Thaveekarn Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand
Jureeporn Noiphrom Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand
Orawan Khow Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand
Sirisak Sathorn Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand

Keywords:

L-amino acid oxidase, King cobra venom, Hydrogen peroxide, Prussian blue, Glutathione

Abstract

L-amino acid oxidase (LAAO) is a flavo-dependent enzyme found in many snake venoms, noted for its cytotoxic and antimicrobial activities through the production of hydrogen peroxide (H₂O₂). This study aimed to develop and evaluate a Prussian blue agar (PBA) assay for LAAO activity in snake venoms, and to purify the enzyme from Ophiophagus hannah, confirming its identity and biochemical characteristics. Venoms from five snake species were screened, and Ophiophagus hannah (King Cobra) venom displayed the highest LAAO activity, resulting in the most prominent blue halo on PBA. This blue coloration resulted from the reaction between H₂O₂ and ferricyanide, leading to the formation of Prussian blue. The halo diameter was quantitatively correlated with known H₂O₂ concentrations. Therefore, it can be used for quantitative assessment. The inclusion of reduced glutathione (GSH), an antioxidant, significantly decreased halo size, illustrating its inhibitory effect on LAAO activity. LAAO was further purified from O. hannah venom through gel filtration, ion-exchange, and affinity chromatography. The identity and purity of the enzyme were verified via SDS-PAGE and protein sequencing. Reverse-phase high-performance liquid chromatography (RP-HPLC) revealed a distinct LAAO peak at approximately 80 minutes, coinciding with the corresponding peak in crude venom. These findings confirmed the presence and retention behavior of LAAO. The PBA method proved to be a rapid, visual, and cost-effective tool for assessing LAAO activity and potential modulators. The PBA method proved to be a rapid, visual, and cost-effective tool for assessing LAAO activity and potential modulators. It holds significant value for applications in toxicology research, enzyme characterization, and the investigation of agents that inhibit its activity.

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