Indoxyl Acetate as a Substrate for Analysis of Lipase Activity.

Tomas Valek,Adam Kostelnik,Miroslav Pohanka,Pavla Valkova

doi:10.1155/2019/8538340

Tomas Valek, Adam Kostelnik + Show 2 more

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https://doi.org/10.1155/2019/8538340

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Abstract

Lipases play a crucial role in metabolism of microbes, fungi, plants, and animals, and in analytical chemistry, they are often used in detection of fats and triglycerides. Determination of lipase activity is also important in toxicology, when lipase activity can be both increased and decreased by organophosphates and other pesticides and in medicine for diagnosis of heart diseases. The standard method for lipase activity determination is based on cleaving ester bonds in lipase buffer containing Tween. Our aim was to find a method with faster and more sensitive response. It is known that acetylcholinesterase belongs to the same group of hydrolases enzymes as lipases and it cleaves indoxyl acetate, so we assume indoxyl acetate could report a similar reaction with lipase. Our method is based on indoxyl acetate as a substrate for lipase, where indoxyl acetate is cleaved by lipase to indoxyl and acetate moiety and blue indigo is created. The method was optimized for different times and amount of enzyme and compared with the standard Tween assay. The calibration curve measured in reaction time 20 minutes with 10 μl of lipase exhibited the best analytical parameters, and it showed Michaelis–Menten response with the Michaelis–Menten constant equal to 8.72 mmol/l. The indoxyl acetate-based method showed faster and more sensitive response than the standard method for lipase activity determination, so it has great potential in biosensor construction and it could be used in industry, medicine, toxicology, and common practice where the activity of lipases is need to be measured.

Highlights

All lipases belong to triacylglycerol hydrolases (E.C. 3.1.1.3) class of enzymes. eir function is to cleave ester bonds in fats, mostly triglycerides, to glycerol and fatty acids
Determination of lipase activity is important in toxicology, when lipase activity can be both increased and decreased by organophosphates and other pesticides and in medicine for diagnosis of heart diseases. e standard method for lipase activity determination is based on cleaving ester bonds in lipase buffer containing Tween
Our method is based on indoxyl acetate as a substrate for lipase, where indoxyl acetate is cleaved by lipase to indoxyl and acetate moiety and blue indigo is created. e method was optimized for different times and amount of enzyme and compared with the standard Tween assay. e calibration curve measured in reaction time 20 minutes with 10 μl of lipase exhibited the best analytical parameters, and it showed Michaelis–Menten response with the Michaelis–Menten constant equal to 8.72 mmol/l. e indoxyl acetate-based method showed faster and more sensitive response than the standard method for lipase activity determination, so it has great potential in biosensor construction and it could be used in industry, medicine, toxicology, and common practice where the activity of lipases is need to be measured

Summary

Introduction

All lipases belong to triacylglycerol hydrolases (E.C. 3.1.1.3) class of enzymes. eir function is to cleave ester bonds in fats, mostly triglycerides, to glycerol and fatty acids. Lipases are known as serine hydrolases because catalytic center contains serine, histidine, and aspartate or glutamate, depending on the type of organism [1,2,3,4]. Importance of lipases has been growing in the medical sector because admission of an external enzyme can even serve as cancer prevention [12]. It appears that lipoprotein lipases with abnormal function correspond with pathological processes like obesity, Alzheimer disease, atherosclerosis, and dyslipideamia associated with diabetes and infections [13]. Organophosphates and carbamates are used in agriculture, medicine, and industry, and they have been developed by chemical manipulation of extremely toxic nerve gasses. eir well-known effect is based on inhibition of acetylcholine esterase (AChE) which leads to disorders of neurotransmission, but they affect the whole metabolism and biochemical pathways [17,18,19]

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