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An Improved End-Point Fluorimetric Procedure for the Determination of Low Amounts of Trypsin Activity in Biological Samples Using Rhodamine-110-Based Substrates

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Abstract

A novel end-point fluorimetric procedure based on the use of rhodamine-110-labeled specific substrate was developed to determine trypsin activities in biological samples. We evaluated the ability of trichloroacetic acid and acetic acid to stop the enzymatic reaction without hindering the detection of the fluorescence of rhodamine-110 released into the reaction mixture from the specific substrate (CBZ-l-alanyl-l-arginine)2-rhodamine-110. Trichloroacetic acid decreased markedly the fluorescence of rhodamine-110, even at low concentrations. On the other hand, the addition of 50 mmol/l acetic acid inactivated efficiently trypsin activity, causing minor effects on rhodamine-110 fluorescence. The proposed procedure was more sensitive than the spectrophotometric end-point method using N-α-benzoyl-dl-arginine-p-nitroanilide as substrate. The possibility of carrying out end-point fluorimetric assays improves the performance of monocell fluorimeters by setting specific conditions optimal for each enzyme activity independently of the fluorimeter. This method also allows replicate assays to be conducted simultaneously, resulting in considerable time saving and in increased performance of low-cost equipment.

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Abbreviations

CBZ:

Benzyloxycarbonyl

TCA:

Trichloroacetic acid

Rho-110:

Rhodamine-110

BPC:

Phosphate-citrate buffer

RFU:

Relative fluorescence units

BAPNA:

N-α-Benzoyl-dl-arginine-p-nitroanilide

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Acknowledgment

This work was funded by the grant AGL2001-1831 from the Spanish Government.

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Authors and Affiliations

  1. Dpto. Biología Aplicada, Universidad de Almería, 04120, Almería, Spain

    Jaime G. Mayoral, Francisco J. Alarcón, Tomás F. Martínez & Pablo Barranco

  2. Department of Biological Sciences, Florida International University, 33199, Miami, FL, USA

    Jaime G. Mayoral & F. Noriega

Authors
  1. Jaime G. Mayoral
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  2. Francisco J. Alarcón
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  3. Tomás F. Martínez
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  4. Pablo Barranco
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  5. F. Noriega
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Correspondence to Francisco J. Alarcón.

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Mayoral, J.G., Alarcón, F.J., Martínez, T.F. et al. An Improved End-Point Fluorimetric Procedure for the Determination of Low Amounts of Trypsin Activity in Biological Samples Using Rhodamine-110-Based Substrates. Appl Biochem Biotechnol 160, 1–8 (2010). https://doi.org/10.1007/s12010-008-8520-9

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  • DOI: https://doi.org/10.1007/s12010-008-8520-9

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