[논문]다양한 감광제와 아미노산의 조합 반응에서 nitro blue tetrazolium의 환원특성 평가

이은빈; 홍정일

doi:10.9721/kjfst.2022.54.1.1

다양한 감광제와 아미노산의 조합 반응에서 nitro blue tetrazolium의 환원특성 평가
Reduction of nitro blue tetrazolium by combined reaction of various photosensitizers with amino acids 원문보기

한국식품과학회지 = Korean journal of food science and technology, v.54 no.1, 2022년, pp.1 - 7

이은빈 (서울여자대학교 자연과학대학 식품응용시스템학부) , 홍정일 (서울여자대학교 자연과학대학 식품응용시스템학부)

초록
AI-Helper

본 연구에서는 형광등 빛 조사 하에 각종 감광제와 아미노산의 조합에 의한 NBT의 환원 특성을 조사하였다. 기존 SOD 활성 측정에 이용된 Rb-Met 반응계는 증류수나 Tris보다는 PBS 용매 상에서 가장 우수한 효과를 나타냈다. 빛 조사 하에 Rb에 의한 NBT 환원을 위해서는 Met과 His 등의 아미노산이 필요하며, 감광제로서는 isoalloxazine 계열의 Rb와 thiazine 계열의 MB가 효과적인 환원 반응을 유도했다. 하지만 각종 감광제나 아미노산의 조합, 그리고 반응 용매에 따라 NBT 환원정도가 상이하며, 특히 Rb-Met in PBS와 MB-His in Tris 반응계가 가장 큰 반응을 유도하였다. Rb에 의해 유도된 NBT 환원반응은 SOD 및 gallic acid에 의해 효과적으로 저해되었으나, Tris 상에서 MB-His에 의한 NBT 환원은 SOD 및 gallic acid에 의한 저해효과가 미미하여 Rb와는 다른 기작에 의해 NBT 환원이 유도되는 것으로 보인다. 본 결과는 감광제와 아미노산에 따라 빛 조사 하에 다양한 상호작용이 발생하며, NBT 환원을 유도하는데 superoxide anion 뿐만 아니라 다른 요인도 관여할 수 있음을 시사한다.

Abstract ▼ AI-Helper

Riboflavin (Rb), in the presence of methionine (Met) under light, generates superoxide radicals that can reduce nitro blue tetrazolium (NBT) to its corresponding formazan. The Rb-Met/NBT system has been used to measure the superoxide dismutase (SOD)-like activities of various antioxidants. However, the reaction mechanisms have not been clearly defined, and the assay conditions are not consistent. In this study, the effects of different photosensitizers and amino acids on NBT reduction in different solvents were investigated. NBT reduction in the Rb-Met/NBT system was more pronounced in phosphate-buffered saline, compared to distilled water or Tris (pH 7.5); histidine (His) instead of Met also led to considerable Rb-induced NBT reduction. Among the photosensitizers, methylene blue with His caused potent NBT reduction in Tris. Rb-induced NBT reduction combined with Met or His was quantitatively inhibited by SOD or gallic acid, but did not affect MB-induced reduction sensitively.

주제어

표/그림 (6)

그림 Fig. 1. Nitro blue tetrazolium (NBT) reduction property by the reaction of riboflavin (Rb)-methionine (Met) in different solvent systems. NBT reduction by different concentrations of Rb with Met (10 mM) was conducted under FL 4000 lx in PBS (A), DW (B), Tris-HCl (C), and in a Dark (D). Each value represents the mean±SD (n=3). Different letters indicate significant difference (p<0.05) based on one way ANOVA and the Tukey's HSD test.
그림 Fig. 2. Nitro blue tetrazolium (NBT) reduction property by the reaction of riboflavin (Rb) combined with different amino acids (methionine (Met), tryptophan (Trp), histidine (His)) in different solvent systems. The reduction properties of NBT by the reaction of Rb with different amino acids were compared at 560 nm in PBS (A), DW (B), and Tris-HCl (C). Each value represents the mean±SD (n=3). Different letters indicate significant difference (p<0.05) based on one way ANOVA and the Tukey's HSD test.
그림 Fig. 3. Nitro blue tetrazolium (NBT) reduction property by the reaction of riboflavin (Rb) with different amino acids (methionine (Met), tryptophan (Trp), histidine (His)) in PBS. The NBT reduction properties by the reaction of different concentrations of Met (A), Trp (B), and His (C) with Rb (4 μM) compared during 2 h in PBS. The extend of NBT reduction at 60 min in each reaction system was also shown (D). Each value represents the mean±SD (n=3). Different letters indicate significant difference (p<0.05) based on one way ANOVA and the Tukey's HSD test.
그림 Fig. 4. The Nitro blue tetrazolium (NBT) reduction property by riboflavin (Rb) or methylene blue (MB) combined with histidine (His) in Tris-HCl buffer. The reduction of NBT using different concentrations of Rb (A) and MB (B) combined with 10 mM of His were evaluated in Tris (pH 7.5). Effect of different concentrations of His on Rb (C) and MB (D)-dependent NBT reduction were also analyzed. Each value represents the mean±SD (n=3). Different letters indicate significant difference (p<0.05) based on one way ANOVA and the Tukey's HSD test.
표 Table 1. The absorbance changes from Nitro blue tetrazolium (NBT) reduction by the combined reaction of different photosensitizers with amino acids in PBS or Tris buffer
그림 Fig. 5. Effects of superoxide dismutase (SOD) and gallic acid on photosensitizer-induced Nitro blue tetrazolium (NBT) reduction. The effects of SOD (A) and gallic acid (B) on NBT reduction induced by 5 combinations of riboflavin (Rb) or methylene blue (MB) with amino acids at 60 min were compared. The effects of SOD (C) and gallic acid (D) on MB-histidine (His)-induced NBT reduction in Tris during 2 h were also shown. Each value represents the mean±SD (n=3), and the numbers in bracket (A and B) indicate correlation coefficient (R²). Different letters indicate significant difference (p<0.05) based on one way ANOVA and the Tukey's HSD test.

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