Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1998-07-02
2000-10-03
Leary, Louise N.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 23, 435 4, 435968, C12Q 137, C12Q 100, G01N 3353
Patent
active
06127139&
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for assaying a proteolytic enzyme comprising the use of a substrate having a fluorescent label and a fluorescence quencher.
Fluorometric assays for proteolytic enzymes, wherein a peptide substrate which is specifically cleaved by the enzyme and which carries a fluorophore at one side and a fluorescence quencher at the other side of the cleavage site are used, are known in the art. For example, EP-A-428000 discloses fluorescence-quenched substrates for detection of viral proteolytic enzymes, the substrates consisting of peptide sequences that can be specifically cleaved by the viral protease, to which are attached a fluorescent donor such as 5-(2-aminoethylamino)naphthalene-1-sulphonic acid (Edans), and a quencher, such as 4-(4-dimethylaminophenylazo)benzoic acid (Dabcyl). Upon contact with the specific proteolytic enzyme, the peptide is cleaved and the fluorescent donor is detached from the quenching acceptor, so that irradiation leads to a detectable fluorescence which is a measure for the specific enzymatic activity. A similar system for the detection of retroviral proteases is disclosed in EP-A-435845. Fluorogenic substrates that can be selectively hydrolysed by matrix metalloproteinase-3 (MMP-3) and to which a substituted coumarin and 2,4-dinitrophenyl are bound as a fluorophore and a quencher respectively, are described by Nagase et al., J. Biol. Chem. 269, 20952-20957 (1994). A general method for the preparation of such internally quenched fluorogenic protease substrates with the Edans/Dabcyl system, is described by Maggiora et al., J. Med. Chem. 35, 3727-3730 (1992).
Although these known substrates and methods allow proteolytic enzymes to be assayed with reasonable accuracy, known substrates and methods with fluorogenic proteinase substrates have been applied with purified enzymes and hardly with complex biological media (e.g. cell culture media, synovial fluid and other tissue fluids). These substrates and methods are likely to be subject to disturbances by fluorescent or light-absorbing constituents of more complex solutions than purified enzymes. According to the invention, a method is provided for quantitatively assaying proteolytic enzymes, using immobilised fluorescence-quenched substrates.
With the immobilized fluorescence-quenched substrates, e.g. on microtiter plates, the fluorescent fragments remain attached to the insoluble carrier after proteolytic cleavage. Thus, disturbing components of the reaction mixture (e.g. blood) can easily be washed away, providing a clean solution for use in the actual measurement. As a result, a more sensitive and reliable assessment of proteolytic enzyme activity is achieved.
Small peptide substrates for proteolytic enzymes suffer from the disadvantage that they may still be hydrolysed by high molecular weight complexes of proteinase and inhibitor, as is the case with matrix metalloproteinases inhibited by .alpha.2-macroglobulin (.alpha.2MG). Inasmuch as MMP-.alpha.2MG complexes are unable to hydrolyse the high molecular-weight physiological substrates such as proteoglycans, plasma proteins and collagen proteins, soluble peptide substrates overestimate the actual proteolytic activity. Immobilised fluorescence-quenched proteinase substrates are not hampered by this disadvantage: as a result of their immobilization, they are not converted any more by MMP-.alpha.2MG complexes. In this event, and all events mentioned below, immobilized is not limited to an insoluble carrier. Also, soluble carriers such as albumin, and macromolecules derived by any means, are applicable. In this context, macromolecules are understood as having a molecular weight of more than 5 kDa, especially more than 10 kDa.
Another aspect of the immobilization procedure is that the carrier (microtiter plate, paper or other strip) can be dried before performing the assay. This offers the advantage that reproducible and convenient assay kits can be developed which require minimal effort of the testing person. More importantly, the "dry format assay" allows asses
REFERENCES:
By H. Nagase, et al., Design and Characterization of a Fluorogenic Substrate Selectively Hydrolyzed by Stromelysin 1 (Matrix Metalloproteinase-3), The Journal of Biological Chemistry, vol. 269, No. 33, Aug. 19, 1994, pp. 20952-20957.
By G.M. McGeehan et al., "Characterization of the Peptide Substrate Specificities of Interstitial Collagenase and 92-kDa Gelatinase", The Journal of Biological Chemistry, vol. 269, No. 52, Dec. 30, 1994, pp. 32814-32820.
Beekman Bob
Te Koppele Johannes Maria
Leary Louise N.
Nederlands Organisatle voor Toegepast-Natuurwetenschappelijk Ond
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