A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

Jorge A Rodriguez,Juan Carlos Mateos-Díaz,Frédéric Carrière,María De Los Angeles Camacho-Ruiz

doi:10.1194/jlr.d052837

Abstract

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidification. Purified lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profiles of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.

Highlights

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator
TG(4:0), TG(8:0), butyric acid (BtA), octanoic acid (OcA), BSA, NaCl, CaCl2, sodium taurodeoxycholate (NaTDC), ␤-cyclodextrin (␤-CD), bromocresol green (BCG), 3,4-dinitrophenol, chlorophenol red, p-nitrophenol, cresol red, thymol blue, malic acid, succinic acid, MES, MOPS, Tris, CHES, 2-methyl-2-propanol, DMSO, and THL were purchased from Sigma-Aldrich
We developed a continuous spectrophotometric lipase assay in microtiter plates using emulsified TG(4:0) and TG(8:0), that are some of the most common substrates used to measure lipase activity, apart from natural longchain TGs

Summary

Introduction

A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsified short- and medium-chain TGs, and a pH indicator. The substrate preference of each enzyme tested was not modified and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples.—Camacho-Ruiz, M. Lipases (TG ester hydrolases, EC 3.1.1.3) are lipolytic carboxylester hydrolases which catalyze the hydrolysis of the ester bonds of TGs to form FFAs and glycerol in some cases They are widely distributed in microorganisms, plants, and animals [1,2,3] where they play an important role in lipid metabolism [4, 5].

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Conclusion