Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Stablizing an enzyme by forming a mixture – an adduct or a...
Reexamination Certificate
1998-11-30
2001-01-23
Woodward, M P (Department: 1631)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Stablizing an enzyme by forming a mixture, an adduct or a...
C435S213000, C435S023000, C424S094300, C424S094640
Reexamination Certificate
active
06177268
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for stabilizing trypsin in which autolysis of trypsin is inhibited, a method for increasing enzymatic activity of trypsin, and to a kit for measuring enzymatic activity of trypsin.
BACKGROUND OF THE INVENTION
When measuring enzymatic activity of trypsin, generally an aqueous solution of trypsin (an enzyme solution), a buffer solution, and a substrate solution are prepared separately, and these three solutions are admixed so as to generate enzyme reaction. When trypsin is dissolved and stored in a purified water, the amount of trypsin often decreases due to autolysis. In order to prevent this phenomenon, the pH of an aqueous trypsin solution is usually adjusted to a range in which the trypsin is inactive but not deactivated. For example, trypsin may be dissolved in a hydrochloric acid aqueous solution (1 mmol/l). Also, in order to further increase the stability of trypsin, calcium ions may be added to the hydrochloric acid aqueous solution.
However, in the above-mentioned conventional method of mixing three solutions, additional labor is required for preparing the solutions and for measuring the enzyme reaction. Furthermore, when measuring the enzymatic activity of trypsin with an automatic analyzer in a clinical test or the like, which requires processing a large amount of sample, it is desired to integrate such a three-solution system into a two-solution system. Therefore, dissolving a substrate in a buffer solution may be considered, but there is a problem that &agr;-benzoyl-arginine-p-nitroanilide (BAPNA) or the like generally used as a synthetic substrate for trypsin is unstable in a buffer solution.
Furthermore, in the above-mentioned automatic analyzer or the like, it is desired to prepare a dry reagent by drying a reagent used for measuring the enzymatic activity of trypsin, and adhering it to a test piece or the like. However, when trypsin is stabilized using hydrochloric acid as mentioned above, the hydrochloric acid volatilizes during the drying step, so that the stability of trypsin is reduced. Although other non-volatile acids may be used in place of hydrochloric acid, there is a possibility that acid concentration is increased in the drying step, resulting in deactivation of trypsin, if such acids are used.
SUMMARY OF THE INVENTION
Accordingly, in order to solve these problems, a new method for stabilizing trypsin, which is different from conventional methods is desired. Furthermore, in a clinical test or the like utilizing enzyme reaction of trypsin, it is desirable to increase the enzymatic activity of trypsin so as to improve test precision, processing speed, and the like. Furthermore, in order to improve efficiency of such a clinical test, an improved kit for measuring the enzymatic activity of trypsin is also desired.
It is a first object of the present invention to provide a method for stabilizing trypsin, in which degradation of trypsin can be prevented, and to provide a stabilized trypsin solution which can be applied to an automatic analyzer. It is further a second object of the present invention to provide a method for increasing the enzymatic activity of trypsin. It is still further a third object of the present invention to provide a kit for measuring enzymatic activity of trypsin, in which enzyme reaction of trypsin can be generated in a two-solution system and degradation of trypsin and its substrate is prevented.
In order to achieve the above-mentioned first and second objects, the present invention provides a method for stabilizing trypsin prior to enzyme reaction or for increasing the enzymatic activity of trypsin, which comprises dissolving trypsin in a buffer solution having a pH at which trypsin is active and containing calcium and/or manganese ions.
Accordingly, in the present invention, trypsin is dissolved in a buffer solution having a pH at which trypsin is active, together with calcium ions or the like as enzymatic activity accelerators. This is contrary to traditional thought. It has been considered that, in such a buffer solution, trypsin attacks against one another causing autolysis, so that the concentration of trypsin is decreased, thus having an adverse effect on stabilization.
Therefore, trypsin has been stored by dissolving it in a hydrochloric acid aqueous solution having a pH range at which it is inactive, while calcium ions as enzymatic activity accelerators have been dissolved in a buffer solution (with a pH at which trypsin is active). However, as a result of elaborate studies and experiments with regard to these conventional conditions for enzyme reaction of trypsin, the inventor of the present invention have found out that, surprisingly, trypsin can be stored in a highly stable condition without causing autolysis, if the trypsin is dissolved in a buffer solution together with calcium ions or the like. Therefore, by employing this method for stabilizing trypsin, it is possible to generate enzyme reaction of trypsin in a two-solution system comprising a buffer solution in which trypsin is dissolved together with calcium ions or the like, and a substrate solution. Accordingly, labor which has been required for preparing for enzyme reaction and for measuring the reaction can be reduced, and also stability of the substrate can be ensured. Furthermore, this method can be applied to an automatic analyzer as well as to drying of reagents, etc.
Furthermore, according to the method of the present invention, enzymatic activity of trypsin can be increased compared to conventional methods.
In the method for stabilizing trypsin or for increasing enzymatic activity of trypsin according to the present invention, the total concentration of calcium ions and manganese ions in the buffer solution is preferably in the range of 1 to 200 mmol/l, particularly preferably 3 to 10 mmol/l. Furthermore, in the case of using calcium ions only, the concentration thereof in the buffer solution is preferably 1 to 200 mmol/l; and in the case of using manganese ions only, the concentration thereof in the buffer solution is preferably 1 to 200 mmol/l. Although there is no difference between calcium ions and manganese ions with respect to the stability of trypsin, because calcium ions are stronger than manganese ions as enzymatic activity accelerators, calcium ions are preferably used. It is also possible to use both of these ions together.
In the method for stabilizing trypsin according to the present invention, the concentration of the buffer solution is preferably at least 10 mmol/l, particularly preferably in the range of 50 to 500 mmol/l.
In the method for stabilizing trypsin according to the present invention, it is preferable that the pKa of the buffer solution is higher than the pH of the buffer solution.
Next, in order to achieve the above-mentioned third object, the present invention provides a kit for measuring enzymatic activity of trypsin, which comprises a trypsin solution in which trypsin is dissolved in a buffer solution having a pH at which trypsin is active and containing at least calcium and/or manganese ions.
In the kit according to the present invention, it is preferable that the kit further comprises a substrate for trypsin as well as the trypsin solution.
In the kit according to the present invention, as in above-mentioned method for stabilizing trypsin, the total concentration of calcium ions and manganese ions in the buffer solution is preferably in the range of 1 to 200 mmol/l, particularly preferably 3 to 10 mmol/l. Furthermore, in the case of using calcium ions only, the concentration thereof in the buffer solution is preferably 1 to 200 mmol/l; and in the case of using manganese ions only, the concentration thereof in the buffer solution is preferably 1 to 200 mmol/l. Although there is no difference between calcium ions and manganese ions with respect to the stability of trypsin, because calcium ions are better enzymatic activity accelerators, calcium ions are preferably used. It is also possible to use both of these ions together.
In the kit according to the present
Arkray Inc.
Merchant & Gould P.C.
Moran Marjorie A.
Woodward M P
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