A spectrophotometric assay for allicin, alliin, and alliinase (alliin lyase) with a chromogenic thiol: reaction of 4-mercaptopyridine with thiosulfinates.
Allicin (diallylthiosulfinate) is the best known active compound of garlic. It is generated upon the interaction of the nonprotein amino acid alliin with the enzyme alliinase (alliin lyase, EC 4.4.1.4). Previously, we described a simple spectrophotometric assay for the determination of allicin and alliinase activity, based on the reaction between 2-nitro-5-thiobenzoate (NTB) and allicin. This reagent is not commercially available and must be synthesized. In this paper we describe the quantitative analysis of alliin and allicin, as well as of alliinase activity with 4-mercaptopyridine (4-MP), a commercially available chromogenic thiol. The assay is based on the reaction of 4-MP (lambda(max)=324nm) with the activated disulfide bond of thiosulfinates -S(O)-S-, forming the mixed disulfide, 4-allylmercaptothiopyridine, which has no absorbance at this region. The structure of 4-allylmercaptothiopyridine was confirmed by mass spectrometry. The method was used for the determination of alliin and allicin concentrations in their pure form as well as of alliin and total thiosulfinates concentrations in crude garlic preparations and garlic-derived products, at micromolar concentrations. The 4-MP assay is an easy, sensitive, fast, noncostly, and highly efficient throughput assay of allicin, alliin, and alliinase in garlic preparations.
Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, Israel.
Talia
T
Miron
Irina
I
Shin
Guy
G
Feigenblat
Lev
L
Weiner
David
D
Mirelman
Meir
M
Wilchek
Aharon
A
Rabinkov
D013437:Carbon-Sulfur Lyases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000031:analogs & derivatives* / Q000032:analysis*; D004220:Disulfides; D005737:Garlic / Q000737:chemistry; D013058:Mass Spectrometry; D009579:Nitrobenzoates / Q000737:chemistry*; D011725:Pyridines / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013438:Sulfhydryl Compounds; D013441:Sulfinic Acids / Q000032:analysis*
D004220:Disulfides; D009579:Nitrobenzoates; D011725:Pyridines; D013438:Sulfhydryl Compounds; D013441:Sulfinic Acids; C011136:thionitrobenzoic acid; C006452:allicin; C042861:4-thiopyridine; C006453:alliin; D013437:Carbon-Sulfur Lyases; C019243:alliin lyase; D003545:Cysteine