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Citation: TIAN Jia-Jia, CHEN Shuang, LI Dong-Hui. Homogeneous Determination of Protease in Acidic, Neutral and Alkaline Media by Fluorescence Anisotropy[J]. Chinese Journal of Applied Chemistry, ;2006, 23(12): 1313-1316. shu

Homogeneous Determination of Protease in Acidic, Neutral and Alkaline Media by Fluorescence Anisotropy

1.
a School of Life Sciences, Xiamen University, Xiamen 361005;
2.
b Cancer Research Center, Medical College, Xiamen University, Xiamen 361005

Corresponding author: LI Dong-Hui,
Received Date: 12 March 2006
Available Online: 6 June 2006
Fund Project: 国家自然科学基金资助项目(20105005) (20105005)福建省自然科学基金项目(F0210012) (F0210012)

This paper reports the application of fluorescence anisotropy technique to the assay of proteases via a protein substrate labeled with tetramethyl isothiocyanate rhodmine(TMRITC).The linear ranges for the assay of pepsin,bromelain and trypsin are 5.0×10^-8~3.0×10^-6,5.0×10^-8~3.0×10^-6,and 3.3×10^-6~3.0×10^-6 g/mL,respectively.The detection limits of the three proteases mentioned above are 26.1×10^-9,14.7×10^-9,5.4×10^-8 g/mL,respectively.The method is suitable for the real-time quantitative and homogeneous determination of a variety of proteases,because the fluorescence behavior of TMRITC is hardly affected in a wide range of pH.The substrate can be prepared easily and is stable under conventional conditions.On the other hand,the emission-wavelength of TMRITC is longer than that of fluorescein isothiocyanate(FITC),a conventional fluorescence probe for tagging proteins,thus it improves the detection of fluorescence anisotropy.In addition,this method is suitable for the determination of protease inhibitors,such as aprotinin,an inhibitor of trypsin.
- fluorescence anisotropy,
- protease,
- rhodamine,
- trypsin,
- pepsin,
- bromelain,
- aprotinin
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