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A general ribonuclease assay using methylene blue

T Greiner-Stoeffele1, M Grunow, U Hahn

  • 1Institut für Biochemie, Fakultät für Biowissenschaften, Pharmazie, und Psychologie, Universität Leipzig, Talstrasse 33, Leipzig, D-04103, Federal Republic of Germany.

Analytical Biochemistry
|August 15, 1996
PubMed
Summary

This study introduces a new spectrophotometric assay for measuring ribonuclease (RNase) activity using methylene blue and RNA. This method enables kinetic studies with the enzyme

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Area of Science:

  • Biochemistry
  • Enzymology

Background:

  • Ribonuclease (RNase) activity is crucial in RNA metabolism and regulation.
  • Current assays often use artificial substrates, limiting physiological relevance.

Purpose of the Study:

  • To develop a novel, direct spectrophotometric assay for quantifying RNase activity.
  • To enable kinetic studies of RNases using their native substrate, RNA.

Main Methods:

  • Assaying RNase activity by monitoring absorbance shifts of methylene blue.
  • Methylene blue intercalates into high-molecular-weight RNA.
  • Spectrophotometric measurement at 688 nm after preincubation with yeast RNA.

Main Results:

  • The assay allows for the direct monitoring of RNA hydrolysis by RNases.

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  • Initial hydrolysis rates can be determined accurately.
  • This method provides a more physiologically relevant approach compared to existing assays.

    • Conclusions:

    • A novel, direct spectrophotometric assay for RNase activity using methylene blue and RNA has been established.
    • This method facilitates enzyme kinetic studies with native RNA substrates.
    • The assay offers a valuable alternative to current methods employing artificial substrates.