《植物生理学报》 2016, 52(7): 1110-1116

白桦悬浮细胞S-亚硝基硫醇比色和荧光测定方法的初步优化

黄雅婷, 菅端, 詹亚光, 范桂枝^*

东北林业大学生命科学学院, 哈尔滨150040

通信作者：范桂枝；E-mail: gzf325@126.com

摘　要：

本文建立了白桦(Betula platyphylla)悬浮细胞中S-亚硝基硫醇含量分析的比色法及荧光检测法。其中, 比色法采用偶氮化反应; 荧光检测法以2,3-二氨基萘(DAN)为探针。比色法结果表明, 供试样品在10~35 min内显色稳定性良好, 重复性高[S-亚硝基硫醇浓度=(7.54±0.11) μmol•L^-1, n=6], 加样回收率为(101.91±2.75)% (n=4); 荧光法结果表明, DAN染色时间和染色浓度均影响白桦悬浮细胞的荧光强度, 其中染色时间为2~4 h和染色浓度在400 μmol•L^-1时细胞荧光强度值最大。由上述结果可知, 比色法测定白桦悬浮细胞中S-亚硝基硫醇含量重复性好, 准确性高, 而荧光检测法直观性强, 建议将两方法结合使用分析白桦等植物细胞中S-亚硝基硫醇含量。

关键词：S-亚硝基硫醇; 比色法; 荧光法; 白桦悬浮细胞

收稿：2016-04-01   修定：2016-06-24

资助：国家自然科学基金(31100445)和中央高校基本科研业务专项资金(2572015DY02)。

Preliminary optimization of colorimetric and fluorometric assays for S-nitrosothiols content determination in birch suspension cells

HUANG Ya-Ting, JIAN Duan, ZHAN Ya-Guang, FAN Gui-Zhi^*

College of Life Sciences, Northeast Forestry University, Harbin 150040, China

Corresponding author: FAN Gui-Zhi; E-mail: gzf325@126.com

Abstract:

In this study, we established colorimetric and fluorometric determinations for S-nitrosothiols content in birch suspension cells. Azo reaction was used in colorimetric method, and 2,3-diaminonaphthalene (DAN) was as a probe in fluorometric assay. Colorimetric assay results indicate that S-nitrosothiols content was (7.54±0.11) μmol•L^-1 (n=6), the rate of sample recovery was (101.91±2.75)% (n=4), and the range of stable time in reagent color was from 10 to 35 minutes. Fluorometric assay shows that DAN dyeing time and dyeing concentrations affected the fluorescence intensity of birch suspension cells, and the maximum fluorescence intensity was under 400 μmol•L^-1 DAN for 2–4 h. In conclusion, the colorimetric method had good repeatability and high accuracy, and the fluorometric method was intuitive. Accordingly, the above two methods are recommended to detect S-nitrosothiols content in plant cells.

Key words: S-nitrosothiols; colorimetric assay; fluorometric assay; birch suspension cells