Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving transferase
Reexamination Certificate
1997-09-17
2001-05-01
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving transferase
C435S007400, C435S007910, C435S193000, C435S810000
Reexamination Certificate
active
06225074
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of detection of enzymatic chemical reactions that result in the cleavage or formation of a chemical, usually covalent bond. Most particularly, the invention is a method for the chromogenic or fluorogenic detection of such enzyme reactions, in particular as an assay screen for new chemical combination that are produced by biotechnology methodologies, that may have activity in enzyme-substrate interactions.
1. Background of the Invention
Enzymes are catalytic proteins that are pervasive in biological systems. Many enzymes catalyze specific reactions which entail the cleavage or formation of a chemical bond. In particular such an Enzyme (E) will increase the rate of reaction of a specific Substrate (S) that involves the formation or cleavage of a covalent bond resulting in a Product (P.) Enzymes are necessary in almost every biological reaction, and helpful in many chemical, pharmaceutical and manufacturing processes. Detecting enzyme activity and defining and measuring enzyme-substrate interactions is desirable in many clinical and laboratory situations, particularly in screening enzyme activity and screening molecules as inhibitors, enhancers or modifiers of pharmacologically interesting enzymes.
2. Description of Related Art
Known enzymes are classified by the International Union of Biochemistry Commission on Enzymes into six distinct categories: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Recent advances in enzymology have identified previously unknown and/or non-natural catalytic molecules that have enzymatic-like speed and specificity, such as extremozymes, abzymes, recombinant enzymes, semi-synthetic enzymes, and catalytic ribozymes.
Recently techniques have been developed which permit large numbers of different chemical compounds to be synthesized rapidly and systematically for drug screening. Large collections of such components called, combinatorial chemical libraries, are expensive to produce so that typically only milligram quantities or less of each different molecule is in a library. Screens for different types of pharmacological or chemical activity can generally require different techniques, different instruments, varying time frames, different sensitivity levels, different software and different methods of data interpretation. As a result, to screen a large combinatorial library, or other large collection of compounds for different types of pharmaceutical or chemical activity heretofore required great expenses for training, instrumentation and reagents. Sifting through such libraries of molecules to determine structural features which show activity and act as possible pharmacological or industrial agents is a tremendous effort.
The field of enzyme study dates back more than one hundred years. Many methods to study and detect enzymatic events are now known. Significantly important enzyme-assay methods include: (1) spectophotometry, using either ultraviolet or visual light; (2) fluorometry; (3) assays involving detection of radioactivity; (4) coupled assays; and (5) enzyme linked immunsorbent assays (ELISA.) Virtually every enzyme requires a specific and unique substrate for its reactivity. The development of an assay for a particular enzyme/substrate reaction is often a difficult endeavor. Because this is a mature field—although still the subject of intense research and development activities—many textbooks and compilations of methods exist, in addition to articles in peer-reviewed journals.
Conventional assays for enzyme activity are virtually as numerous as the number of enzymes. Some examples of conventional assays for enzyme activity are:
creatine kinase, which is used as a serum control for the diagnosis of muscle deterioration is most frequently assayed in a coupled system with pyruvate kinase and lactate dehydrogenase;
proteases are conventionally measured using specific synthetic substrates which contain a chromogenic or fluorogenic enzyme conjugate at the amide bond which is hydrolyzed by the enzyme;
chloramphenicol acetyl transferase (CAT) is a widely used reporter gene in expression studies. There are several commercially available assays. Such assays for CAT include enzyme linked assays (ELISA), radioactive assays and fluorescent methods. Conventional ELISA methods for assaying for CAT typically take from 2-4 hours and are generally sensitive to only 1-2.5×10
−12
g/ml of enzyme. The radioactive and fluorescent assays use expensive and/or dangerous reagents, and typically require a time-consuming post-event separation to measure the CAT activity.
Due to ease of use, specificity and sensitivity, the current method of choice for detection is most assay systems is radioactivity. However the rapid decay of the radioactive probe, danger of radiation exposure, extensive processing of samples, and storage and disposal problems for radioactive materials make non-radioactive methods of detection desirable.
For conventional non-radioactive detection systems to work, synthetic substrates must be designed to report on the event being monitored. In the case of many proteases, hydrolysis products serve to report on the activity of the protease. However, such hydrolysis products are frequently carcinogenic.
The design of non-radioactive methods usually involves the attachment of chromophores, fluorophores or lumigens at the scissile bond of the reporter substrate. A signal is generated if the enzymatic event takes place because a detectable chromogenic, fluorogenic or lumigenic species is liberated. Designing such reporter substrates is difficult; when a probe is introduced onto the substrate, the substrate can lose its lock and key fit to the enzyme, thus losing its enzyme specificity. If the substrate still fits the enzyme, the binding and energetics of the enzyme catalysis may be altered in significant ways with the result that the synthetic reporter substrate will not be a true measure of the enzyme reaction.
Proteases are enzymes that hydrolyze proteins. All living organisms contain proteases to metabolize proteins, regulate cellular processes, defend themselves against exogenous proteins and mediate other important requirements of survival. In the purification of any protein from a natural source, it is necessary to inhibit endogenous proteases to prevent them from breaking down the proteins of interest. The search for the presence of known and unknown proteases in a sample is an important endeavor both at the research stage and the development stage in all areas of biotechnology and related sciences. If the protease is known and it is known to be in the sample of interest, methods for its analysis and strategies for its purification or inhibition will generally have been elaborated. If a sample contains a protease which is not known or, which is not known to be present in that sample, it may be difficult to determine that one is even present in that sample and considerably more difficult to determine what type of protease it is in order to inhibit its activity.
Proteases are classified into a number of categories dependent upon their mechanism of action. They are further classified dependent upon the cleavage site in the proteins which they hydrolyze. The combination of action and cleavage site leads to multiplicative complexity in determining what proteases are present and how their activities might be inhibited. There is no single cleavage site in a protein which will be hydrolyzed by all enzymes and therefore no known substrate which will be a reporter for all enzymes. More importantly, there is no rapid, sensitive high-throughput method to characterize many types of enzyme activity in a single experiment.
Existing art is aimed at determining if any protease activity is present in a sample and is limited to two types of tests:
a. Fluorescently labeled casein;
b. Electrophoretic mobility assay.
Each of these assays typically requires lengthy incubation, is therefore slow (the test may take hours). Moreover the assays are generally insensitive, and may
Phelps David J.
Wright Dennis
Kalow & Springut LLP
Weber Jon P.
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