Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1990-03-08
1993-06-22
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
435 74, 435975, C12Q 100, C12Q 168
Patent
active
052216063
DESCRIPTION:
BRIEF SUMMARY
This invention relates to enzyme assays and substrates for use in such assays.
The presence or absence of certain enzymes in vivo is a useful indicator of illness or deficiency in the organism concerned. Enzymes are also useful in monitoring microbial growth in fermenters and in the food industry and are important in enzyme-linked immunosorbent assays (ELISA), and in the characterisation of bacterial species in culture.
A method of assaying enzymes is to prepare a substrate for each enzyme labelled with a releasable chromogenic group which is colourless or of pale colour when attached to the substrate, but which is strongly coloured when released by enzyme attack on the substrate (i.e. the reagent is of the form S--Z where S is the structural element recognised by the enzyme and Z is the chromogenic group released by enzymic cleavage of the S--Z bond).
U.K. Patent 2008103 described the use of certain nitrovinylphenyl derivatives for this purpose. However such nitrovinylphenyl derivatives suffer a number of disadvantages:
(i) Lack of sufficient water solubility. Usually with some of these nitrovinylphenyl substrates one cannot exceed 2 millimolar solution, and in the case of carboxylic acid esters of nitrovinylphenols, the solubility is very much less. Consequently, it is rarely possible to achieve sufficient solubility of the substrate to saturate the enzymes to be detected. Higher solubilities can be achieved in solvents such as methanol and dimethylsulphoxide, but enzymes are generally adversely affected by such solvents.
(ii) When the nitrovinylphenyl substrates need to be incorporated into kit form for easy and convenient use, they must be supplied as ready-made solutions, because of the difficulties associated with solubilising these substrates. This gives rise to problems of stability and a preferred method of presentation would be as a readily water-soluble solid, preferably in admixture with buffer salts, from which the user could conveniently make up the required substrate solution.
(iii) To develop fully the colour of the chromogenic nitrovinylphenol group, basic conditions are required, preferably pH 9.5. At such pH's, some chemical breakdown of the substrate is possible leading to the release of the chromogenic group, producing an undesirably high blank value to be offset.
(iv) At such high values of pH, nitrovinylphenols can undergo a Michael reaction or a reverse Nef reaction, leading to rapid fading of the generated colour and, at pH 9.5, experience has shown that the final readings have to be taken preferably within 5 minutes.
The applicants have aimed to overcome these disadvantages by providing enzyme substrates which are more water soluble than the nitrovinylphenol substrates, capable of releasing highly coloured chromogenic groups, the full colour of which is generated as close to neutral pH as possible, and is not susceptible to chemical breakdown in ordinary usage. In addition, substrates which afford insoluble chromogens are useful in some circumstances where the released chromogen is collected on a filter paper or membrane, or is strongly absorbed in such a way as to be water-fast. The colour of filter paper or membrane is then an indicator of the presence or absence of the particular enzyme (e.g. ELISA assays). This particular property is valuable in "dipstick" devices in which immobilisation of the chromogenic phenol is important in order to achieve an even colour.
According to this invention a reagent for enzyme assay comprises a substrate consonant with a given enzyme to be assayed, labelled with a chromogenic group which, on action of the given enzyme, is liberated to give a coloured product, characterised in that the chromogenic group has the general formula: Het is any heterocyclic group capable of extending the delocalisation of the electrons from the aryl group. X is NR or O, where R is H, alkyl or any suitable group that will not hinder the enzyme. Enzymes which may be assayed by the use of the appropriate substrates, include aryl esterases, carboxyesterases, lipases, acid
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Praill Percy F.
Price Robert G.
Richardson Anthony C.
Smith Brian V.
King's College London
Meller Mike
Robinson Douglas W.
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