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Molecules 2018, 23(9), 2204;

A Simple Method for On-Gel Detection of Myrosinase Activity

Department of Botany, Division of Pharmacognosy, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary
Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, Sóstói str. 31/b, H-4400 Nyíregyháza, Hungary
Department of Organic Chemistry, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary
Author to whom correspondence should be addressed.
Received: 5 July 2018 / Revised: 27 August 2018 / Accepted: 28 August 2018 / Published: 31 August 2018
(This article belongs to the Special Issue Analysis of Peptides and Proteins by Electrophoretic Techniques)
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Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4 is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification. View Full-Text
Keywords: myrosinase; thioglucosidase; sulfatase; on-gel detection; desulfo-sinigrin; LC-ESI-MS myrosinase; thioglucosidase; sulfatase; on-gel detection; desulfo-sinigrin; LC-ESI-MS

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Gonda, S.; Szűcs, Z.; Plaszkó, T.; Cziáky, Z.; Kiss-Szikszai, A.; Vasas, G.; M-Hamvas, M. A Simple Method for On-Gel Detection of Myrosinase Activity. Molecules 2018, 23, 2204.

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