The invention relates to an enzyme detection method and to its use in detection of parasites.
Protozoan parasites have an adverse effect on the health of human and animal populations in a large number of countries. Problems presented by protozoan parasites are particularly of concern in tropical areas of the world where modern diagnostic methods are often not easily accessible.
Nucleoside hydrolases are enzymes which hydrolyse nucleosides between the ribose or deoxyribose and the purine or pyrimidine base groups. A number of different types of nucleoside hydrolases are known. N-Ribohydrolases hydrolyse ribonucleosides and N-deoxyribohydrolases hydrolyse deoxyribonucleosides. Within each of these two general groups of enzymes are enzymes of differing specificities.
Among the well-characterized N-ribohydrolases, specificity is high for the ribosyl group but varies for the leaving group purine or pyrimidine. The inosine-uridine nucleoside hydrolase (IU-nucleoside hydrolase) from the trypanosome Crithidia fasciculata hydrolyzes all of the naturally occurring purine and pyrimidine nucleosides with similar catalytic efficiencies. The guanosine-inosine enzyme (GI-nucleoside hydrolase) from the same organism has a strong preference for the eponymous substrates and is nearly inert with the pyrimidine nucleosides1,2,3,4. AMP Nucleosidase from bacterial sources is highly specific for the adenine base, and the 5xe2x80x2-phosphoryl is required for significant hydrolytic rates5,6. Nucleoside phosphorylases have a similar mechanism to nucleoside hydrolases and, for example, purine nucleoside phosphorylase is specific for inosine and guanosine substrates and activates phosphate or arsenate anions to attack C1 of the nucleosides.
It is an object of the invention to provide a method of detecting and/or assaying for the presence of certain enzymes, especially those of parasites in samples taken from parasitised humans and animals.
In one aspect the invention provides a method of detecting and/or assaying nucleoside hydrolases using a chromogenic substrate.
Preferably the chromogenic substrates have the formula: 
where X is OH or H, and Y is the residue of Yxe2x80x94OH where Yxe2x80x94OH is a chromophore or a compound readily converted to a chromophore and the substrates are hydrolysed by the nucleoside hydrolase to yield ribose or 2-deoxyribose plus Yxe2x80x94OH.
Preferably the chromogenic substrates are of Formula I wherein X and Y are as defined and the substrates are phosphorylysed by the nucleoside phosphorylase to yield ribose-1-phosphate or 2-deoxyribose-1-phosphate plus Yxe2x80x94OH.
Y may be chosen so that Yxe2x80x94OH is a compound absorbing in the visible or UV light, readily measured at wavelengths greater than 300 nm, preferably greater than 340 nm.
Preferably Yxe2x80x94OH is 6-hydroxynicotinamide or 2-hydroxypyridine-4-carboxamide.
Y may be chosen so that Yxe2x80x94OH is a chemiluminescent compound eg luminol which when released and oxidized by chemical or enzymatic means emits light.
Alternatively Yxe2x80x94OH may be a compound readily converted to a coloured compound eg by reaction with a diazonium salt eg xcex1-naphthol.
More preferably Yxe2x80x94OH is a fluorescent compound eg 4-methylumbelliferone or fluoroscein.
Most preferably Yxe2x80x94OH is a coloured compound eg phenolphthalein, p-nitrophenol, thymolphthalein, 2-nitrophenol, 2-hydroxy-5-nitropyridine.
It is preferred that Yxe2x80x94OH can be measured in the presence of the compound of Formula I by virtue of Yxe2x80x94OH absorbing or fluorescing to a greater extent than the compound of Formula I at certain wavelengths. Generally this will be by virtue of Yxe2x80x94OH ionising and being in equilibrium with quinonoid-like forms.
A particularly preferred substrate is p-nitrophenyl xcex2-D-ribofuranoside. Also particularly preferred is 4-pyridyl xcex2-D-ribofuranoside. Further particularly preferred substrates are 4-methylumbelliferyl xcex2-D-ribofuranoside (4-methylcoumarin-7-yl xcex2-D-ribofuranoside) and 2-(5-nitropandyl) xcex2-D-ribofuranoside.
According to another aspect of the invention, there is provided a method for detecting and/or assaying for parasites especially protozoa in samples obtained usually from humans or animals using a chromogenic substrate, preferably of Formula I.
More preferably the chromogenic substrate is one where Yxe2x80x94OH is fluorescent or coloured.
Another preferred type of substrate is one where Yxe2x80x94OH is chemiluminescent eg luminol.
According to a further aspect of the invention there is provided a kit containing materials for detection or assay of hydrolysis of the chromogenic substrate, preferably of Formula I, by enzymes in a biological sample. Preferred kits comprise the chromogenic substrate in dry form, together with a buffer. Other components eg a cell-lysing agent may also be included.
According to a further aspect of the invention there is provided a dipstick containing a chromogenic substrate, preferably of Formula I, for use in detecting nucleoside hydrolases.
According to a further aspect of the invention there are provided novel compounds of the invention of formula I. In this aspect of the invention X and Y are as previously defined except the known compounds xcex1-naphthyl xcex2-D-ribofuranoside, 4-methylcoumarin-7-yl 2-deoxy-xcex2-D-ribofuranoside, p-nitrophenyl xcex2-D-ribofuranoside, p-aminophenyl xcex2-D-ribofuranoside, 5-amino-6-chloro-3-pyridazinyl xcex2-D-ribofuranoside, 4-chlorophenyl xcex2-D-ribofuranoside, phenyl xcex2-D-ribofuranoside, 4-methoxyphenyl xcex2-D-ribofuranoside, 4-hydroxyphenyl xcex2-D-ribofuranoside, 4-(N,N,N-trimethylammonio)phenyl xcex2-D-ribofuranoside, 4-acetylphenyl xcex2-D-ribofuranoside and the xcex2-D-ribofuranosides of L-DOPA and L-xcex1-methyl-DOPA and the N-acetyl methyl esters of the abovementioned DOPA derivatives are not included in this aspect. The exceptions are not known as chromogenic substrates or for use in assay of parasites. Generally the chromogenic group is not coloured when present in the compound of Formula I but is readily detectable when that compound is hydrolysed to give Yxe2x80x94OH.
Preferred compounds are those defined above in which Y is an optionally substituted pyridyl group or a nitrophenyl group.
Particularly preferred novel compounds of the invention include 3-trifluoroacetamidophenyl xcex2-D-ribofuranoside, 3-aminophenyl-xcex2-D-ribofuranoside, 1-tetralone-5-yl xcex2-D-ribofuranoside, 3-(4-hydroxyphenyl)-1(3H)isobenzofuranone-3-(phen-4-yl) xcex2-D-ribofuranoside, 2-nitrophenyl xcex2-D-ribofuranoside, 4-methylcoumarin-7-yl xcex2-D-ribofuranoside, 3-pyridyl xcex2-D-ribofuranoside, 4-pyridyl xcex2-D-ribofuranoside, 2-(5-nitropyridyl) xcex2-D-ribofuranoside, 5-quinolyl xcex2-D-ribofuranoside, the xcex2-D-ribofuranoside of luminol, p-nitrophenyl 2-deoxy-xcex2-D-erythro-pentofuranoside, 3-carboxamido-6-pyridyl xcex2-D-ribofuranoside, and 4-forymylphenyl xcex2-D-ribofuranoside.
In a further aspect of the invention there is provided a method of preparing a chromogenic substrate of the invention.
According to a further aspect the invention may be directed to a method to detect or assay for the presence of nucleoside phosphorylases using a chromogenic substrate.