Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving oxidoreductase
Patent
1991-11-12
1994-03-29
Brown, Johnnie R.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving oxidoreductase
435190, 435122, 435189, C12Q 132, C12N 904
Patent
active
052984141
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to two new enzymes isolated from microorganisms, the microorganisms which produce these enzymes, the use of the enzymes in catalysing the degradation of heroin and a method and apparatus for the detection of heroin using these enzymes.
2. Description of the Prior Art
There is an urgent need for a method of detection of heroin in particulate form and in body fluids. In relation to particulate heroin, although many different analytical systems have been proposed, most are based on large pieces of equipment, such as mass spectrometry. In relation to body fluids, immunoassay has been used but this is better suited to laboratory, testing where operatives with specialist skills are available. In principle, a cheaper and easier method for heroin detection could be provided if an enzyme specific for heroin was available and if the products of the enzymatic reaction could be detected using relatively simple instrumentation. Unfortunately, the range of enzyme activities presently available is rather limited and these enzymes do not possess the specificity required.
SUMMARY OF THE INVENTION
We have now found two enzymes which can be used in the detection of heroin (3,6-diacetylmorphine). One is an acetylmorphine carboxylesterase (hereinafter "AMCE") which catalyses the hydrolysis of heroin and has a high specificity for heroin. The other is a morphine dehydrogenase ("MDH") which has a high specificity for morphine, which is produced by hydrolysis of heroin. Either of these enzymes, or the two in combination, can be used to detect heroin, by reacting the enzyme with heroin or morphine obtained by its hydrolysis, and detecting the occurrence of an enzyme-catalysed reaction. One such method of detection of the reaction is electrical. In one electrical method, the reaction of heroin with the AMCE liberates acetate ions, which are detectable conductimetrically. The reaction of morphine with morphine dehydrogenase (MDH) requires a cofactor such as nicotinamide adenine dinucleotide phosphate (NADP.sup.+), which is reduced to NADPH concurrently with the oxidation of morphine to morphinone by the enzyme. This redox reaction can be used to generate an electric current at an electrode surface, particularly with the help of a redox mediator. Accordingly, the invention includes electrical sensors, especially of the conductimetric type for the AMCE alone and of the amperometric type for the AMCE and MDH enzymes together. These and other sensors, which can be of an optical, potentiometric, thermal or piezoelectric type, for example, for in the basis of convenient, portable sensors for detecting grains of heroin in the body fluids, luggage, clothing etc. of smugglers, traffickers and heroin users. Accordingly, this invention provides an important advance in the fight against and control of use of drugs.
The term "heroin" used throughout the specification comprises the free base and salts thereof, unless the context requires a more specific meaning.
In a first aspect, the invention provides the acetyl esterase enzyme. Since it catalyses the hydrolysis of heroin (3,6-diacetylmorphine), 3-acetylmorphine and 6-acetylmorphine, it is conveniently termed an acetylmorphine carboxylase (AMCE). It is thus distinguished from the commonly available porcine liver carboxylesterase which displays little or no activity towards the 6-acetyl group of heroin. The ability of the AMCE of the invention to act on the 6-acetyl group is an important distinguishing characteristic. Another distinctive feature of the AMCE is that it has a molecular weight of about 200,000 Daltons (as determined by elution from a gel filtration column calibrated with marker proteins). By the term "about" we mean to encompass variations which are usual in the determination of high molecular weights by this method and certainly to include a variation of up to 10%. Such a high native molecular weight of 200,000 is different from that of other acetyl carboxylesterases; those described for the nocardiof
REFERENCES:
patent: 4204044 (1980-05-01), Suhara et al.
patent: 4916069 (1990-04-01), Fujiwara et al.
patent: 4927759 (1990-05-01), Schofield et al.
R. Ammom et al. Chemical Abstracts vol. 100(3) No. 17087q (1984).
F. Crespi et. al. Chemical Abstracts vol. 110(5) No. 33279r (1989).
J. A. Owen et. al. Chemical Abstracts vol. 99(19) No. 151702a (1983).
D. A. Smith et. al. Chemical Abstracts vol. 85(3) No. 13602c (1976).
P. Liras et. al. Chemical Abstracts vol. 84(5) No. 27348r (1976).
V. Jhaveri. Chemical Abstracts vol. 89(3) No. 18999e (1978).
J. of Biol. Chem., vol. 260, No. 9 pp. 5265-5270. Yamano et al. (1985).
CA 100:17087q; Derout (1984).
Bruce Neil C.
Lowe Christopher R.
Stephens Lauren D. G.
British Technology Group Limited
Brown Johnnie R.
Varma A.
LandOfFree
Detection of morphine using morphine dehydrogenase does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Detection of morphine using morphine dehydrogenase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Detection of morphine using morphine dehydrogenase will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-790945