Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 4 to 5 amino acid residues in defined sequence
Patent
1992-06-11
1994-08-02
Cashion, Jr., Merrell C.
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
4 to 5 amino acid residues in defined sequence
435 23, 435 18, C07K 500, C07K 510
Patent
active
053347030
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a new chromogenic synthetic substrate used for quantitative determination of bacterial endotoxins in physiological fluids, food, pharmaceuticals etc.
THE BACKGROUND OF THE INVENTION
Bacterial endotoxins are produced by Gram negative bacteria and are considered by most investigators to be a very important factor in the development of septecemia. Several methods for determination of endotoxin have been described based on the observation by Levin and Bang (1956)that endotoxins specifically activate the clotting system of Limulus Potyphemus. In the beginning a test, in which Limulus Amebocyte Lysate (LAL)in contact with endotoxins containing sources, produce a specific gelation, had been developed. More recently chromogenic and fluorogenic methods based on the above observation have been developed and can rapidly detect small amounts of endotoxins (H. C. Hemker: Handbook of Synthetic Substrates, 1983, Martinus Nijhoff Publisher, Boston).
PRIOR ART
A. E. Torano et al have disclosed that an enzyme from Limulus amebocyte lysate shows similar specificity to mammalian blood coagulation factor X.sub.a (Thrombosis Research 34, 407-417, 1984), which recognize the sequence -Ile-Glu-Gly-Arg- in its natural substrate prothrombin.
Other investigations have also Shown the important role the COOH-terminal Gly-Arg sequence plays when analysing endotoxin. T. Harada et al, Biomedical Applications of the Horseshoe Crab (Limulidal), E. Cohen (ed), Alan R. Liss Inc. New York, 1979, pages 209-220, disclose in a table on page 213 different substrates which are used, but only those having the carboxy termial sequence Gly-Arg give interesting results. On page 212, lines 7-10 from the bottom, the authors point out: "These results clearly indicate that Limulus clotting enzyme displays a high specificity towards the peptide pNA having COOH-terminal Gly-Arg sequence".
U.S. Pat. Nos. 4,188,264 and 4,576,745 also give Gly-Arg as carboxy-terminal sequence in the substrate used for determination of endotoxin. Other substrates have been investigated, all of them showing Arg as carboxy terminal.
According to U.S. Pat. No. 4,406,832 the carboxy terminal should be -Ala-Arg- or -Cys-Arg-and this substrate has given relative activity which is as good as or a little better than standard -Gly-Arg-. Due to the complicated mechanism in these reactions it is not possible to know which peptide sequence could give an acceptable result.
Surprisingly and against the prior art within the field, we have now found that a substrate which has the carboxy terminal sequence -Gly-Lys- gives a relative activity which is at least 20% better compared with known substrates.
The use of Glycolic acid (Glyc) in a substrate for determination of endotoxin has never been disclosed before, and it is very surprising that the effect is as good when using -Glyc-Arg- as when using Gly-Lys, both of them giving a better effect than the normally used Gly-Arg.
DESCRIPTION OF THE INVENTION
The chromogenic synthetic peptide or peptide isostere derivative in the present invention shows high sensitivity in the method used for the determination of endotoxins.
The new substrates are characterized by the following formula: is Glyc when A.sub.4 is Arg.
The present invention also discloses the process for the preparation of the peptid derivatives.
It also discloses the method for determination of bacterial endotoxins by the use of the derivative with the following formula: which permit quantifying of endotoxins by determination of splitted marker directly or after derivatization (H. C. Hemker: Loc. cit.) with the proviso that A.sub.3 is Gly when A.sub.4 is Lys and that A.sub.3 is Glyc when A.sub.4 is Arg.
The use of these derivatives for determination of bacterial endotoxins is also disclosed.
Example of compounds which could be R.sub.2 -NH-.sub.2 are: p-nitroaniline, 3-carboxy-4-hydroxyaniline, 3-sulfo-4-nitroaniline, 3-alkoxy-4-nitroaniline, 3-carboxy-4-nitroaniline, 4-methyloxy-naphtylamine, 4-(N-ethyl-N-hydroxyethyl) amino
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Cashion Jr. Merrell C.
Chromonogenix AB
Salata Carol A.
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