A Rapid and Precise Method to Determine Cysteine Content in Food Materials using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

Abstract

A method to determine cysteine content in food materials using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxidiazole (ABD-F) was compared to a method using N-acridinyl maleimide (NAM). Cysteine in samples was derivatized with two fluorogenic reagents and analyzed using HPLC. The cysteine derivative with ABD-F gave a single peak while the cysteine derivative with NAM produced three peaks in standard. The cysteine content in model samples determined with ABD-F was nearly identical to the content determined with NAM. However, when the cysteine content of six types of food materials was measured, four samples were identical in each method, but two samples produced different results because the separation of cysteine derivatives with NAM was influenced by other compounds in the food samples. Since the ABD-F method gave the better separation, it can be useful for the determination of cysteine content in food materials.Keywords: 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxidiazole (ABD-F), N-acridinylmaleimide (NAM), cysteine *To whom correspondence should be addressed. E-mail: hiroaki_nishiuchi@ajinomoto.com IntroductionL-Cysteine, one of the 20 natural amino acids, is an important sulfur-containing compound because it is the only source of sulfur for cell metabolism in most organisms. Cysteine is indispensable for protein folding, protein assembly, and protein stability through disulfide bond formation with the reactive thiol residue. In addition to this metabolic importance, cysteine also plays an important industrial role. Cysteine has been used for industrial applications such as pharmaceuticals, cosmetics, bakery, and flavorings, resulting in consumption of approximately 4000 tons/y globally (Wada et al., 2006). For example, cysteine was reported to be effective as a reducing agent in production of French bread, crackers, and cookies (Grandvoinnet et al., 1979). In the beverage industry, cysteine was effective in preventing browning of fruit juice, such as grape juice or pear juice, during concentration (Skalski and Sistrunk, 1974; Montgomery, 1983.) In addition, cysteine prevented the development of off-flavor in stored orange juice . In flavor chemistry, cysteine is an important source of sulfur in a variety of aromas (Strakenmann et al., 2008). Thus, cysteine will continue to play an important role in the food industry. Therefore, a reliable method to determine the cysteine content in food materials is necessary.Several methods have been developed to determine cysteine content. Previously, mercaptide-forming reactions using metal ions were used. However, more recent methods use fluorescent reagents to react with thiol residues. For example, NAM, a maleimide reagent, provides blue fluorescence by reaction with thiol compounds and has been used for analysis of thiol compounds such as cysteine or glutathione (Nara et al., 1978;Takahashi et al., 1979;Meguro et al., 1996). In addition, NAM has been used to determine cysteine content in commercial yeast extracts (Kuroda et al., 1997). In contrast, ABD-F, a reagent with a halo...

An Improved Method to Determine Gamma-glutamylcysteine Content in Foodstuffs Using 4-(Aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

“…Similarly, the specificity, linearity, and precision of this method were demonstrated for the determination of GSH content in model samples. Moreover γ-GC and GSH derivatives were as stable as cysteine derivatives at 4℃, the stability of which was previously reported (Nishiuchi et al, 2011). Therefore, this improved method permitted the simultaneous determination of γ-GC and GSH contents in many foodstuffs.…”

“…Determination of γ-GC and GSH contents by HPLC using ABD-F HPLC analysis and derivatization using ABD-F were performed as per a previous report on cysteine determination (Nishiuchi et al, 2011). A standard compound or sample was dissolved in 5 mM sodium acetate buffer containing 2 mM EDTA (pH 4.0) at appropriate concentrations.…”

“…Therefore, it is important to have a rapid and precise method to determine the γ-GC content in foodstuffs. In this study, we improved the method to determine cysteine content by using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F) (Nishiuchi et al, 2011), which is suitable for γ-GC determination. In addition, we used this improved method to measure the γ-GC content in several foodstuffs in an effort to elucidate their potential sources.…”

Recent advances in development and application of derivatization reagents having a benzofurazan structure: a brief overview

“…. ABD‐F (4‐aminosulfonyl‐7‐fluoro‐2,1,3‐benzoxadiazole), SBD‐F (ammonium 7‐fluoro‐2,1,3‐benzoxadiazole‐4‐sulfonate) and DBD‐F were used for the derivatization of thiols such as cysteine, homocysteine, N ‐acetylcysteine, methionine and glutathione (Huang et al ., ; Nishiuchi et al ., ; Otani et al ., ; Ferin et al ., ; Wada et al ., ; Isokawa et al ., ). Isokawa et al .…”

“…1. ABD-F (4-aminosulfonyl-7-fluoro-2,1,3-benzoxadiazole), SBD-F (ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate) and DBD-F were used for the derivatization of thiols such as cysteine, homocysteine, N-acetylcysteine, methionine and glutathione Nishiuchi et al, 2011;Otani et al, 2011;Ferin et al, 2012;Wada et al, 2013;Isokawa et al, 2013). Isokawa et al (2013) used hydrophilic interaction chromatography for the separation of SBD-thiols.…”