CS248348B1 - Method for determining enzyme activity - Google Patents

Abstract

The solution relates to a method for determining enzymes, in particular alkaline and acid phosphatase, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase, which consists in reacting a sample containing the enzyme under investigation with a substrate which, depending on the enzymatic activity, releases substances with a free thiol group, while when determining the activities of enzymes that displace groups other than thiol, for example amino groups, hydrogen and others, substances with a free thiol group are released by subsequent reactions with additional enzymes, the course of their release is monitored by an electrochemical reaction. The method of determination can be used in medicine.

Description

The invention relates to a method for determining the activity of enzymes of medical interest, in particular alkaline phosphatase - ALP /E.C. 3.1.3 ·! / And acid phosphatases - ACP /E.C. 3.1.3.2./, aspartate aminotransferase - AST /E.C. 2.6.1.1./ and alanine aminotransferases - ALT /E.C. 2.6.1.2./, both formerly called GO and GP transaminases, and also lactate dehydrogenases - ID /E.C. 1.1.1.27 · and others.

Methods for determining alkaline and acidic phosphatase activities today are based on the cleavage of a substrate - eg, nitrophenyl phosphate - whose cleaved portion is colored, eg, nitrophenol, and color development and its photometry is a measure of enzyme activity. Aspartate aminotransferase and alanine aminotransferase were determined by imperfect methods with visible light photometry (Reitman method, Frankel), but this method, due to its imperfection, is abandoned and replaced by continuous ultraviolet photometry at 340 nm or fluorometer. The essence is the redox changes of nicotinamidadenine dinucleotide, which results in changes in absorbance. Thus, this reaction is indicative and is often accomplished by a chain of further reactions and the addition of other enzymes and substances. Naturally, this procedure requires the possibility of continuous photometry and its registration in ultra violet light, which is relatively costly.

These deficiencies are eliminated by a method for determining the activity of enzymes, in particular alkaline and acidic phosphatases, aspartate aminotransferases, alanine aminotransferases and lactate dehydrogenases according to the invention, characterized in that the sample containing the enzyme of interest acts on a substrate which releases substances with free thiol group. whose release is monitored by an electrochemical reaction. When determining the activities of enzymes that move non-thiol groups,

248,348 w

For example, amino groups, hydrogen and others, the free thiol groups are liberated by subsequent reactions with additional enzymes, for example, coenzyme A is liberated from acetyl coenzyme A by addition of citrate synthase to determine aspartate aminotransferase activity, or by addition of pyruvate carboxylase and citrate synthase. The oxygen-depleted substrate is treated with a sample containing the enzyme of interest, followed by a voltage potential trace between the free thiol group and the calomel electrode, and the enzymatic activity of the sample is determined from the angle difference between the blank and enzyme line. .

The present method is based on the possibility of electrochemical continuous detection of the resulting thio groups either directly by cleavage of the substrate, or by subsequent reactions with additional enzymes, using the apparatus described in cs. No. 134022.

For phosphatases, a suitable substrate is, for example, the S-phenylthiophosphate lithium salt (or S- (p-methoxyphenyl) thiophosphate (aniline salt)), where enzymatic cleavage at an appropriate pH results in the cleavage of the phosphate residues having a free thiol group. Consequently, their concentration increases continuously and a linear recording of the phosphatase enzyme activity occurs on the electronic compensation recorder.

The method of determining the enzymatic activity of the invention is illustrated in more detail by way of examples.

Example 1

The following agents are used in the process of the invention: carbonate-bicarbonate buffer 0.1 M, pH 10.0 for alkaline phosphatase or sodium citrate with 0.1 M hydrochloric acid buffer, pH 4.9 for acid phosphatase, substrate S- / p-methoxyphenyl (thiophosphate / aniline salt) at a concentration of 2.10 ' - * M dissolved in the appropriate buffer, followed by pressurized nitrogen in the bottle.

4 ml of buffer and 0.5 ml of substrate solution are dispensed into the vial and purged for 2 min. nitrogen and bring the mixture to 50 ° C; drop the mercury drop electrode with a drop time of about 3 seconds and record a blank test on the recorder. Then add

- 3,248,348

0.2 ml of enzyme sample, purged again with nitrogen for 20 seconds and then recorded the linear course of enzymatic activity. The blind line angle is a measure of phosphatase activity. Calibrate with 1 millimolar reduced glutathione.

Example 2

To measure aspartate aminotransferase - AST activity, a conventional substrate is used: L-aspartate and 2-oxoglutarate in a suitable buffer, but citrate synthase / E.C. is added again. 4 · 1 · 3 · 7 / and acetyl coenzyme A. Then the reaction proceeds as follows:

Λςφ

L-aspartate + 2-oxoglutarate ->

, Oxalacetate citrate synthase acetyl coenzyme A ci-13-σ

S.CoA oxalacetate + L-glutamate (1) citrate coenzyme A

CoA.SH

With the formation of citrate, coenzyme A is formed, which has a free thiol group and a linear record of the course of the enzymatic activity of AST occurs.

The following reagents are used in the process of the invention: substrate / containing 0.1 M in 100 ml phosphate or other buffer, pH 7.8: 17 mmol of L-aspartate, 1.5 mmol of 2-oxoglutarate, 2.5 mg of citrate synthase - about 400 units, 0.01 mmol acetyl coenzyme A / and nitrogen compressed in the bottle.

4 ml of the substrate is dispensed into the vial and purged for 2 min. nitrogen and warm the solution to 30 ° C; drop the mercury drop electrode with a drop time of about 3 seconds and record the blank test. 0.2 ml of the aspartate aminotransferase sample is then added, the mixture is purged again with nitrogen for 20 seconds, and then the linear course of the enzymatic activity is recorded. The blind line angle is a measure of aspartataminotransferase activity. Calibrate with 1 millimolar reduced glutathione.

Example 3

To measure the activity of alanine aminotransferase - ALT, a common substrate is used: L-alanine and 2-oxoglutarate in a suitable buffer, but

248 348 further add pyruvate carboxylase / E.C. 6.4.1.1/, citratesyntha for /E.C. 4.1.5.7/ and acetyl coenzyme A. Then the reaction proceeds as follows:

L-alanine + 2-oxoglutarate

ALT pyruvate - oxaloacetate acetyl coenzyme A pyruvate carboxylase

pyruvate + L-glutamate oxalacetate citrate coenzyme A / 1 / / 2 /

Again, the formation of citrate also produces coenzyme A, which has a free thiol group and produces a linear notation of the course of enzymatic activity of alanine aminotransferase.

The following reagents are used in the process of the invention: substrate / containing 0.1 M in 100 ml phosphate or other buffer, pH 7.8: 40 mmol of L-alanine, 1.5 mmol of 2-oxoglutarate, 1.5 mg of pyruvate carboxylase about 10 units, 0.15 mM adenosine triphosphate,

2.5 mg citrate synthase - about 400 units, 0.01 mmol acetyl coenzyme A / and nitrogen compressed in the bottle.

4 ml of the substrate is dispensed into the vial and purged for 2 min. The mercury drop electrode is dropped with a drop time of about 5 seconds and a blank is recorded. Then 0.2 ml of serum is added, the mixture is again purged with nitrogen for 20 seconds, and then a linear course is recorded. enzymatic activities. The blind line angle is a measure of alanine aminotransferase activity. Calibrate with 1 millimolar reduced glutathione.

Example 4

To measure lactate dehydrogenase - LD activity, a common substrate is used: L-lactate and NAD ⁺ in a suitable buffer and newly added pyruvate carboxylase (EC 6.4.1.1) and citrate synthase (EC 4.1.5.7) and acetyl coenzyme A. Then the reaction proceeds as follows:

L-Lactate + NAD ⁺ _ ... ¾-- ru pyruvate + NADI-I + H + / 1 / pyruvate oxalacetate pH 9 - 9,5 pyruvate carboxylase ^ citrate synthase oxalacetate citrate / 2 / · acetyl coenzyme A coenzyme A

- 5 248 348

With the formation of citrate, coenzyme A, which has a free thiol group, is formed and a linear record of the enzymatic activity of lactate dehydrogenase is formed.

The following reagents are used in the process of the invention: substrate / containing 0.1 M in 100 ml of carbonate or other buffer, pH 9.3: 0.1 mmol of L-lactate, 0.02 mmol of NAD-nicotinamide adenine dinuclide, 5 mg pyruvate carboxylase - about 10 units, 0.13 mmol adenosine triphosphate, 2.5 mg citrate synthase - about 400 units, 0.01 mmol acetyl coenzyme A / and nitrogen compressed in the bottle.

4 ml of the substrate is dispensed into the vial and purged for 2 min. nitrogen and bring the solution to 30 ° C; drop the mercury drop electrode with a drop time of about 3 seconds and record the blank test. 0.2 ml of the enzyme sample is then added, the mixture is purged again with nitrogen for 20 seconds, and then the linear course of the enzymatic activity is recorded. The angle of the blind line is a measure of lactate dehydrogenase activity. Calibrate with 1 millimolar reduced glutathione.

This makes use of a simple and inexpensive device to measure the activity of the described clinically important enzymes in serum, which is otherwise only possible with the costly device for continuous photometry in ultraviolet light.

Claims (3)

Method for determining the activity of enzymes, in particular alkaline and acidic phosphatase, aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, characterized in that a sample containing the enzyme of interest is treated with a substrate which releases substances with free thiol group depending on enzymatic activity. by the activity of enzymes that displace other than thiol groups, for example amino, hydrogen and others, the free thiol group substances are released by sequential reactions with additional enzymes, whereupon the release progress is monitored electrochemically. 2. The method of claim 1, wherein the oxygen-depleting substrate is treated with a sample containing the enzyme of interest, followed by monitoring the voltage potential profile between the free thiol group and the calomel electrode and the difference in angle between the blank and the blank. the enzymatic activity of the sample is determined corresponding to the enzyme sample. 5. A method according to claim 1 or 2, characterized in that in determining the aspartate aminotransferase activity, coenzyme A is released from acetyl coenzyme A by addition of citrate synthase. 4. A method according to claim 1 or 2, characterized in that in determining the activity of alanine aminotransferase or lactate dehydrogenase, coenzyme A is liberated from acetyl coenzyme A by addition of pyruvate carboxylase or citrate synthase.