Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1996-04-24
1999-04-27
Tsang, Cecilia J.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
530415, C12Q 148
Patent
active
058980695
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the detection and quantification of inorganic phosphate, especially in biological solutions. More particularly, the present invention relates to a modified phosphate binding protein and the use of such a protein in a phosphate assay.
In biological systems, changes in phosphorylation state and fluctuations in the concentration of inorganic phosphate are associated with a large number of important events. For example, a number of diseases and conditions present with elevated or depressed levels of serum inorganic phosphate concentration. Moreover, the major energy requirements of the body are fulfilled by deriving energy from alterations in the phosphorylation state of nucleotides.
Because inorganic phosphate (P.sub.i) is involved in a large number of important biological processes, it is desirable to be able to measure the concentration of Pi and the changes in such concentration in biological systems Phosphate assays, which measure Pi concentration, are useful in a number of diagnostic methods, as well as in research into the functioning of biological systems.
To date, two classes of phosphate assay have been described. These are enzymatic inorganic phosphate assays and chemical inorganic phosphate assays. See, for example, Lindberg and Ernster, 1956, and Veldhoven and Mannaerts, 1987.
Enzymatic phosphate assays, for example as described in European Patent Application 0 159 513, are based on a phosphate-requiring enzyme, often a phosphorylase. In the method described in EP 0 159 513, a purine-nucleoside phosphorylase in used to convert a nucleoside (inosine) to ribose-1-phosphate and a base, in this case hypoxanthine. Hypoxanthine is then converted into a coloured agent, from which the extent of inosine conversion, which is dependent upon P.sub.i concentration, may be determined.
Another assay based on a purine nucleoside phosphorylase is described in Webb, 1992a.
Enzymatic phosphate assays tend to be relatively insensitive For example, the method described by Webb may not be used below concentrations of 2 micromolar P.sub.i. Furthermore, although more rapid than chemical phosphate assays, enzymatic phosphate assays remain too slow to allow study of the kinetics of many biological systems.
Chemical phosphate assays, for example as described by Veldhoven and Mannaerts (1987), rely on the chemical generation of colour upon exposure to phosphate. For example, many chemical methods are based on the complex formation of phosphomolybdate at low pH with basic dyes, which causes a shift in absorption maxima of these dyes. The colour change is measured by spectrophotometry.
Chemical methods have the disadvantage of being extremely slow, although great sensitivity may be achieved. For example, the method of Veldhoven and Mannaerts is claimed to be sensitive to 1.5 micromolar P.sub.i, to detect picomole quantities of P.sub.i.
The universality of phosphate in biological systems, and the extremely high incidence of phosphate contamination in laboratory solutions makes the development of phosphate assays sensitive beyond the micromolar range of concentrations relatively unimportant. However, it remains desirable to be able to produce phosphate assays having an extremely rapid response rate, in order to monitor the kinetics of biological and chemical processes which involve the production or consumption of P.sub.i.
A number of proteins are known which specifically bind to P.sub.i. For example, transport of P.sub.i into and out of cells and organelles is executed by specific transport proteins. In bacterial cells, it is achieved by way of a high affinity transport system dependent on a phosphate-binding protein. Such proteins are able to specifically recognise inorganic phosphate, bind to it and transport it across cell membranes or between cellular compartments.
An example of such a protein is the E. coli phosphate binding protein (PBP) which is encoded by the phosgene of E. coli. This protein is located in the periplasm of E. coli as part of the P.sub.i scavenging system of the bacterium
REFERENCES:
Richieri et al., "A Fluorescently Labeled Intestinal Fatty Acid Binding Protein", J. Biol. Chem., vol. 267, No. 33, pp. 23495-23501, Nov. 25, 1992.
Morita, Biosis #84;169742 (ABS only), 1983.
Richieri, Biosis #93:70384 (ABS only), 1992.
Bakin, Caplus #1988:565902 (ABS only), 1988.
Nag, Caplus #1990:548234 (ABS only), 1990.
Brune Martin Hermann
Corrie John Edgar Thomas
Webb Martin Ronald
Delaney Patrick R.
Medical Research Council
Powers Vincent M.
Tsang Cecilia J.
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