Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1995-07-20
2001-05-08
Lee, Howard C. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S018700, C536S020000, C536S021000, C536S029100, C536S122000, C536S123000, C536S123100
Reexamination Certificate
active
06228998
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a novel derivative of a glycosaminoglycan in which an allyl compound is bonded to a reducing end sugar of a glycosaminoglycan via an aminoalkyl bond or an acid amide bond. It further relates to a glycosaminoglycan-bonded polyacrylamide gel in which the above-mentioned novel derivative is copolymerized with acrylamide. It furthermore relates to a process for identifying a glycosaminoglycan-degrading enzyme using the above-mentioned polyacrylamide gel as a gel electrophoresis carrier.
BACKGROUND OF THE INVENTION
There have been known glycosaminoglycan-bonded substances formed by binding various substances (for example, proteins, phospholipids, lipids and the like) to a hemiacetal of a reducing end sugar of a glycosaminoglycan optionally activated. Attempts have been made to apply these substances to drugs (JP-A-3-284698 corresponding to EP-A-454898, International Publication No. WO92/01720 corresponding to EP-A-493622; the term “JP-A” as used herein means an “unexamined published Japanese patent application”).
Also, slight changes in the amount or microstructure of glycosaminoglycans have attracted public attention with regard to the infection with bacteria or viruses, cancer, hereditary diseases, etc. These changes frequently depend on glycosaminoglycan-degrading enzymes occurring in a trace amount in cells, tissues and body fluids. Accordingly, it has been regarded as important to assay these enzymes.
It is not suitable for assaying glycosaminoglycan-degrading enzymes to use such a method as those employed in assaying glycosidase, i.e., one comprising using a substrate consisting of a monosaccharide or an oligosaccharide and a chromogenic compound or a fluorescent compound bonded thereto and observing and measuring the rate and the manner of appearance or disappearance of the color development (or fluorescence) due to the digestion with the enzymes. Thus, these enzymes have been assayed by using a glycosaminoglycan as a substrate and determining the disaccharide or oligosaccharide formed by digestion or examining a decrease in the molecular weight. There has been known a gel electrophoretic method for assaying a protease or the like which is called “zymography” and comprises electrophoresing an enzyme on a gel having a substrate uniformly embedded therein and, after the digestion of the substrate with the enzyme, measuring the loss in the substrate. In this method, however, high molecular weight compounds are exclusively usable as the substrate, since a low molecular weight substrate per se would be electrophoresed and thus flow out from the gel. Moreover, these methods are disadvantageous in that a complicated operation is required, sensitivity and reproducibility are poor and applicable substrates are limited.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a process for identifying a glycosaminoglycan-degrading enzyme which is free from the above-mentioned problems. The present invention also provides a novel glycosaminoglycan derivative which is useful as a substrate in the above-mentioned process as well as an electrophoresis gel carrier composed of a copolymer of this novel derivative and acrylamide.
Under these circumstances, the present inventors have conducted extensive studies in order to easily and accurately assay a trace amount of a glycosaminoglycan-degrading enzyme with a high sensitivity, and as a result, the present invention has been completed.
Accordingly, the gist of the present invention resides in: 1) a glycosaminoglycan derivative in which a compound having an allyl group at one end which is not concerned in bonding with a glycosaminoglycan is bonded to a reducing end sugar of said glycosaminoglycan via an aminoalkyl bond or an acid amide bond, more specifically said reducing end sugar is an aldehyde group formed by reducing and partially oxidizing the reducing end sugar of a glycosaminoglycan, or a lactone formed by oxidizing and cyclodehydrating the reducing end sugar of a glycosaminoglycan, or a glycosaminoglycan derivative in which a spacer compound having at least two amino groups is bonded to an aldehyde group or a lactone formed as described above via an aminoalkyl bond or an acid amide bond, respectively, and further a hydrocarbon compound having an allyl group at one end and a functional group capable of binding to an amino group at another end and having or not a heteroatom in the chain, is bonded to the amino group of said spacer compound; 2) a glycosaminoglycan-bonded polyacrylamide gel which is a copolymer composed of the glycosaminoglycan derivative as described above and acrylamide as constituting monomers; and 3) a process for the identification of a glycosaminoglycan-degrading enzyme which comprises electrophoresing a specimen containing the glycosaminoglycan-degrading enzyme by using the glycosaminoglycan-bonded polyacrylamide gel as described above as a carrier, after the electrophoresis, incubating the carrier under such conditions as to proceed the enzymatic reaction, and then detecting the degradation of the glycosaminoglycan fixed to the carrier to thereby separate and identify the glycosaminoglycan-degrading enzyme in the specimen.
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Miura et a., “Analysis of Glycosaminoglycan-Degrading Enzymes by Substrate Gel Electrophoresis (Zymography)”, Anaytical Biochemistry, vol. 225, No. 2, pp. 333-340, Mar. 1995.*
Raja et al., “Preparation of Alkylamine and125I-Radiolabeled Derivatives of Hyaluronic Acid Uniquely Modified at the Reducing End”, Analytical Biochemistry, vol. 139, pp. 168-177, 1984.*
Fiszer-Szafarz, “Hyaluronidase Polymorphism Detected by Polyacrylamide Gel Electrophoresis. Application to Hyaluronidases from Bacteria, Slime Molds, Bee and Snake Venoms, Bovine Testes, Rat Liver Lysosomes, and Human Serum”,Analytical Biochemistry, 143:76-81 (1984).
Laemmli, “Cleavage of STructural Proteins Proteins During the Assembly of the Head of Bacteriophage T4”,Nature, 227:680-685 (1970).
Steiner et al, “A Zymographic Assay for Detection of Hyaluronidase Activity on Polyacrylamide Gels and Its Application to Enzymatic Activity Found in Bacteria”,Analytical Biochemistry, 200:405-410 (1992).
Guntenhöner et al, “A Substrate-Gel Assay for Hyaluronidase Activity”,Matrix, 12:388-396 (1992).
Miura et al, “Analysis of Glycosaminoglycan-Degrading Enzymes by Substrate Gel Electrophoresis (Zymography)”,Analytical Biochemistry, 225:333-340 (1995).
Miura Ryu
Yamagata Sadako
Yamagata Tatsuya
Lee Howard C.
Seikagaku Kogyo Kabushiki Kaisha
Sughrue Mion Zinn Macpeak & Seas, PLLC
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