Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2001-03-05
2002-02-26
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C435S018000, C562S125000
Reexamination Certificate
active
06350586
ABSTRACT:
BACKGROUND OF THE INVENTION
Uncontrolled tissue destruction by protease release plays a role in some pathological conditions, and it is hypothesized that an imbalance in the protease antiprotease system is an important contributing factor. Elastase is a protease that is capable of hydrolyzing the connective tissue component elastin. Elastase acts on a protein's nonterminal bonds adjacent to an aliphatic amino acid. Elastase is one of the matrix degrading enzymes found in plasma and is closely associated with elastin tissue destruction found in conditions such as abdominal aortic aneurysm (AAA) (Cohn, J. R., et al.
Surg. Gynecol. Obstet
. 1987; 164:355-358); emphysema (Snider, G. L., et al.
Am. J. Resp. Crit. Case. Med
. 1994; 150:5131-5137); pancreatitis (Dominguez-Muneoz, J. E., et al.
Dig. Dis. Sci
. 1993; 38:507-513; Stein, J., et al.
Clin. Chem
. 1996; 42:222-226); and inflammatory conditions (Andus, T., et al.
Dig. Dis. Sci
. 1993; 38:1638-1644), such as arthritis.
Since abdominal aortic aneurysms often go unnoticed until they become large enough to be felt as a mass or to cause symptoms, a method for the early diagnosis of an abdominal aortic aneurism is needed, and wound be extremely useful for early treatment of aneurysms.
In order to understand the role of elastase in these conditions one must be able to determine elastase activity. There are a variety of ways to quantitatively measure proteases, such as elastase, such as the use of radiolabeled elastin (Bieger, W., and Sheele, G. (1980)
Anal. Biochem
. 104, 239-246), second enzyme-coupled elastin (Saunders, G. C., Svitra, Z., and Martinez, A. (1982)
Anal. Biochem
. 126, 122-130), and synthetic substrates (Gertler, A., and Hofmann, T. (1970)
Can. J. Biochem
. 48, 384-386; Bieth, J., Spiers, B., and Wermuth, C. G. (1974)
Biochem. Med
. 11, 350-357; Zhou, D., Niewiarowski, S., and Stewart, G. J. (1995)
Anal. Biochem
. 224, 436-437), radioimmunoassay (Murata, A., Ogawa, M., Fugimoto, K-I, Kitahara, T., Matsuda, Y., and Kosaki, G. (1983)
Enzyme
30, 29-37), zymography (Brophy, C. M., Sumpio, B., Reilly, J. M., and Tilson, D. M. (1991)
Surg. Res. Commun
. 10, 315-321), and ELISA (Dreher, M., Gunzer, R., Helger, R., and Lang, H. (1989)
Prog. Clin. Biol. Res
. 308, 707-710). However, these techniques have limitations, such as low sensitivity, they are time-consuming and labor-intensive, and they are unable to distinguish between elastase and zymogen (inactive enzyme). There is therefore an outstanding need for a rapid and sensitive method of detecting and measuring elastase in a sample for use in the understanding of the role of elastase in connective tissue disorders and for diagnosing connective tissue disorders.
Habeeb, A.F.S.A.,
Anal. Biochem
. 14:328-336 (1966) describes a method of detecting the presence of free amino groups in proteins using 2,4,6-trinitrobenzenesulfonic acid. Hatakeyama, et al.
Anal. Biochem
. 204:181-184 (1992) describe an assay for detecting protease using succinylcasein as a substrate. Bubnis and Ofner,
Anal. Biochem
. 207:129-133 (1992) describe the detection of &egr;-amino groups in proteinaceous material using trinitrobenzenesulfonic acid. These references do not, however, address the need for a method of determining specific inhibitors of elastase.
The role of elastase in connective tissue disorders may be better understood through the identification of inhibitors of elastase, since the loss of inhibition of elastase is an important contributing factor for the development of connective tissue disorders. Elastase may be inactivated by naturally occurring inhibitors that block the active site of the enzyme by binding tightly to it. These naturally occurring elastase inhibitors are necessary to prevent elastase from unnecessarily degrading proteins which can lead to connective tissue destruction. Once inhibitors of elastase are identified, they may be used in the design of drugs for treating these connective tissue disorders. Thus, an outstanding need remains for a method of detecting inhibitors of proteases, such as elastase, for the development of inhibitors for diagnosing and treating connective tissue disorders.
SUMMARY OF THE INVENTION
The present invention provides an assay for detecting and quantifying protease enzymes that is sensitive, rapid, and capable of screening numerous samples of small volume. The method provided by the present invention specifically detects and quantifies elastase in a biological sample by contacting the biological sample with a succinylated protease substrate, adding to the biological sample containing the substrate an amount of trinitrobenzenesulfonic acid (TNBSA) sufficient to detect the presence of primary amines in the sample, and measuring the amount of the primary amines. The amount of the primary amines in the sample is indicative of the amount of protease present therein.
Also provided by the present invention is a method for diagnosing disorders associated with connective tissue destruction in a subject by detecting and quantifying a particular protease in a biological sample.
The present invention provides a method of detecting inhibitors of the activity of a protease by detecting primary amines that are exposed by the enzymatic degradation of a protease substrate. Generally, the inhibitory effect of a substance on the activity of a protease is determined by introducing the substance into a reaction mixture containing a succinylated protease substrate, and the reaction is initiated by the addition of a known amount of a protease. The inhibitory effect of the substance is then evaluated by comparing the activity of the protease in the presence and absence of the substance.
The method of the present invention may be used to detect proteases such as elastase, other elastolytic enzymes, and trypsin by using substrates such as elastin, casein and gelatin. The method of detection may be used to detect proteases in biological samples including, but not limited to, whole blood, plasma, serum, synovial fluid, urine, saliva, sputum, mucous secretions, cell culture supernatant, tissue biopsies, and tissue homogenates.
Additional objects of the invention will be apparent from the description which follows.
REFERENCES:
patent: 6017723 (2000-01-01), Rao et al.
patent: 6197537 (2001-03-01), Rao et al.
Cohen Jon R.
Kushner Leslie
Mathrubutham Mahesh
Rao Srinivasa K.
Amster Rothstein & Ebenstein
Leary Louise N.
Long Island Jewish Medical Center
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