Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Patent
1997-11-05
1999-12-28
Lankford, Jr., Leon B.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
435 25, 435 28, 435189, 435190, 435192, 435219, C12N 900, C12Q 137
Patent
active
06008006&
DESCRIPTION:
BRIEF SUMMARY
This invention relates to the determination of glycated proteins; more particularly, it relates to such a method involving the use of a proteinase in the same reagent as a peroxidase and to a kit therefor.
Horseradish peroxidase is an oxidoreductase (donor:hydrogen peroxide oxidoreductase; EC 1.11.1.7). It is widely used in the life sciences as an indicator enzyme (see, for example, Essays in Biochemistry, 1994; 28: 129-146), and is one of a family of peroxidase enzymes. The particularly useful features of this enzyme are its ease of coupling to carriers, such as antibodies or other enzymes, its high rate of activity with a range of substrates and good thermal stability. It consists of a single polypeptide comprising 308 amino acids and has a relative molecular mass of 44,000, which incorporates a haemin prosthetic group giving it a brown colouration. The enzyme has four disulphide bridges and contains two calcium ions, removal of which leads to a reduction in stability.
This enzyme catalyses the transfer of hydrogen from a hydrogen donor to a hydrogen acceptor. The hydrogen acceptor is usually hydrogen peroxide, although methyl and ethyl peroxides may also be used. Hydrogen peroxide is reduced according to the following reaction:
A wide range of hydrogen donors may be used. These include phenols, aminophenols, indophenols, diamines and leuco dyes. The oxidative process of hydrogen removal from such compounds generates products which may be detected visually or quantified, usually in a spectrophotometer. Other means of detection used have included fluorimetry, luminometry and electrochemistry.
The enzyme may be physically coupled to other proteins, such as antibodies or fragments thereof. This allows the specific binding properties of the antibody to be used to measure an analyte or to identify histologically the location of an antigen. It may also be chemically linked to an oxidase enzyme to quantify the substrate of the oxidase. There are many analytes that may be measured using specific oxidases. Of these, several are present in biological fluids where analysis thereof may be clinically helpful. The use of phenol and aminoantipyrene as chromogens linked to an oxidase--peroxidase system has long been known (see, for example, Ann Clin Biochem, 1969, 6: 24-27). More recently, alternatives to phenol, such as N-ethyl-N-(2-hydroxy-3-sulphopropyl)-m-toluidine (TOOS), have been proposed which are more sensitive and are coloured over a wide pH range (see, for example, Chem Pharm Bull., 1982; 30: 2492-2497).
One such analyte is glycated protein, or fructosamine. This is the product of a non-enzymatic reaction by which glucose or other sugars may form condensation products with free amine groups of protein (see, for example, Clin Chem, 1987; 33: 2153-2163. In the blood, the main proteins that are glycated are albumin, in which exposed lysine residues provide the free amine group, and haemoglobin, in which the N-terminal valine amino acid may also react with glucose. In diabetic subjects, the concentrations of the protein components of blood vary between fairly narrow limits. In contrast, the glucose concentration may change significantly in a short period of time. Many of the pathological changes experienced by diabetic patients are the consequence of prolonged exposure of proteins to elevated glucose concentrations. Therefore, the measurement of glycated protein is clinically useful in assessing the average glucose exposure over the lifetime of the protein.
Several methods have been used to measure total glycated protein. The current reference method is the furosine procedure (see, for example, J Clin Chem Clin Biochem, 1981; 19: 81-87). This involves protein digestion in 6 molar hydrochloric acid at 95-100.degree. C. for 18 hours. Furosine is a product of glycated lysine under these conditions and may be measured by HPLC. This method is too complex and time-consuming for routine use. The thiobarbituric acid procedure is slightly simpler as it uses a shorter acid digestion (2-5 hours) yielding 5-hydroxymethylfurf
REFERENCES:
patent: 5370990 (1994-12-01), Staniford et al.
Poller Sarah Catherine
Shipley Darren Paul
Torrens David John
Genzyme Limited et al.
Lankford , Jr. Leon B.
Salcedo F. Brad
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