FR2610409A1 - METHOD AND AGENT FOR THE DETECTION OF THIOL GROUPS - Google Patents

Abstract

THE PRESENT INVENTION CONCERNS THE DETECTION OF THIOL GROUPS PRESENT IN A MEDIUM OR WELL FORMED DURING A STEP PRECEDING THE DETECTION REACTION. THIS FORMATION MAY BE CARRIED OUT BY CHEMICAL REACTIONS SUCH AS, FOR EXAMPLE, REDUCTION OF DISULPHIDES, OR BIOCHEMICAL REACTIONS SUCH AS, FOR EXAMPLE, HYDROLYSIS OF THIOESTERS BY ESTERASES. FE IONS ARE REDUCED TO FE IONS BY THIOL GROUPS. THE FE IONS ARE THEN DETECTED BY MEANS OF SUITABLE COMPLEX LIGANDS. APPLICATION: PERFECTED METHOD AND AGENT FOR THE DETECTION OF THIOL GROUPS.

Description

The subject of the present invention is a method and an agent for

  detection of thiol groups present

  in a medium or trained in a previous step

  the detection reaction. This formation can be carried out by chemical reactions such as, for example, the reduction of disulfides, or by

  biochemical reactions. The preferred biochemical reactions

  are illustrated by the following equation:

Liponamide-

  dehydrogenase a) CONH2 _'_ _ _ _ _ _ CCNH2

SH SH

43 of

NADH + H NAD

  CHe [3 Cholinesterase b) CH3-1-CH2CH2-S-C-CH3 CH3 CH3

LCH3-N-CHZCH2-SH + CH3COOH

  Many methods are known for the detection of compounds containing thiol groups. A particularly useful and frequently mentioned method is that described by GK Ellman in "Arch Biochem Biophys 1959, 82, 70-77", which is based on the formation of the yellow anion of 3-mercapto-6-acid. -nitrobenzoic acid, which is formed by reaction between a thiol and the acid

  3,3'-dithio-bis-6-nitrobenzoic (Ellman's reagent).

  A major disadvantage of Ellman reagent is the only obtaining of yellow color shades by

2610 4 0 9

  reduced disulfides. However, nuances of color

  ration ranging from red to blue would be desirable for

the visual determination of the thiol groups.

  Specifically, these color shades can be developed by the method according to the present invention. The present invention is based on the reaction

following fundamental: -

  2 R-SH + 2 Fe 3 ± R-S-S-R + 2 Fe 2 + Iron at oxidation state 3 is reduced by iron thiol groups at oxidation state 2, the thiol groups being themselves oxidized to disulfides. The formed Fe2 + ion is then converted by suitable ligands into complexes, the molecular extinction coefficient of which is a multiple of the molecular extinction coefficient.

3+ 2+

  Fa complexes. In addition. Fe complexes have a shift compared to Fe complexes, so

  that a clear variation of color can be observed.

  The subject of the present invention is an analytical agent for the detection of thiol groups which contains Fe3 ions which can be reduced to 2+ Fe ions by thiol groups and a complex ligand which is capable of forming a colored complex with the ions. Fe forms. The detection of Fe 2 ions by means of suitable complex ligands in the form of colored complexes is known. Examples of suitable ligands for this purpose are compounds selected from the group consisting of

  ferroines, cuproins and terroines.

  Complex ligands such as hydrazones and their tautomeric azo forms, tetrazolylpyridines, pyridylquinazolines, bisisoquinolines, imines, phenanthrolines, bipyridines, terpyridines,

  bidiazines, pyridyldiazines, pyridylbenzimidines,

  azoles, diazyltriazines, orthonitroanilines, phenols, tetrazines, triazines, pyridines, phenazine, quinoxalines, benzimidazoles,

  oximes of methyl- or phenyl-2-pyridyl-ketones sub-

  stituées, are particularly suitable (Smith, Analyt.

  Chem., 26 (1954) 1534-1538; Schilt et al., Talanta (1968) 475-478; Schilt et al., Talanta (1968) 1055-1058; and Schilt et al.

Talanta 16 (1969) 448-452.

  A description of other complex ligands is

  found in Blandamer et al., J. Chem. Soc. Dalton (1978), 1001-1008, Case, J. Org. Chem., 31 (1966), 2398-2400, as well as in British patent application N 701 843. However, complex ligands other than

  those mentioned there can also be used.

  For the use of the analytical agent and therefore also complex ligands in analytical strips, it may be advantageous for the complex ligands to also carry hydrophobic radicals or functions acting as ion exchangers. The binding of the complex ligands to the matrix is thus improved and

  the "exudation" of the test strip is thus avoided.

  The hydrophobic radicals that can be used are long-chain alkyl or aralkyl radicals. The binding of complex ligands to polymers is also

conceivable.

  The analytical agent according to the present invention can be used for the detection of thiols such as the amide of liponic acid, thiocholine,

  glutathione or coenzyme A, or thioglycosides.

  The thiol may also be present as a precursor in the form of a thioester, a thioether, a disulphide

  or a thioacetal in the analytical agent.

  Thus, the analytical agent according to the present invention is also suitable, for example, for the detection of enzymes which cleave thio compounds. In this case, there may be mentioned thioether hydrolases, thioesterases or thioglycosidases. However, esterases such as

  cholinesterase (CHE) can also be detected.

  The natural substrate of CHE is acetylcholine. However, this enzyme is also able to cleave acetyl and butyrylthiocholine. The free thiol group formed by the

  cleavage is then detected by means of the analytical agent.

  Biochemical reactions in which free thiol groups are formed via

  Redox reactions are furthermore known.

  We can mention in this case the reduction

  of liponic acid catalyzed by liponamide-dehydro-

  genase in the presence of nicotinamide adenine dinucleotide

reduced (NADH).

  Accordingly, it is also possible to use the analytical agent according to the present invention in the analysis of NADH dependent reactions. Typical examples of NADH-dependent enzymes that may be mentioned are:

  lactate dehydrogenase, alcohol dehydrogenase, glucose

  dehydrogenase, glyceraldehyde dehydrogenase, glycerolphosphate dehydrogenase, malate dehydrogenase, etc. NADH can also be the end product of multi-step enzymatic reactions, as in the case of glutamate-oxalo-acetate-transaminase (EC 2. 6.11), glutamate-pyruvate-transaminase

  (EC 2.6.12) or creatine-quinase (EC 2.7.32).

  NADH can also be the reaction product in the analysis of substrates such as lactate, glucose, malate, urea, etc. By the use of the analytical agent according to the present invention in the analysis of NADH-dependent reactions, a notable improvement in sensitivity is achieved, in particular due to the formation of two moles of Fe 2 per mole of NADH and very high extinction coefficients presented

  in some cases by the colored complexes.

  It has been found to be advantageous to provide the Fe 3 comulex bound to the thiol group substrate. Possible complexing agents are EDTA, HEDTA, citric acid, malic acid, lactic acid, amino acids such as, for example, alanine, glycine and glutamine, ethers. crown configuration such

  that, for example, an 18-crown-6, phenylaza-15-

  crown-5, benzo-15-crown-5 and dibenzopyridino-15-crown-5, or triazinophanes and cryptates, and oxidized sugars such as, for example,

  for example, gluconic acid and glucuronic acid.

  The ratio of thiol: Fe3 concentrations should be at least 1: 1 and preferably 1: 5. The ratio 1:20 is particularly appreciated for the reaction

  as fast as possible, and the 1:10 ratio is particularly

advantageously.

  It should be understood that the analytical background or analytical composition, in the context of the present invention, refers, for example, to those which can be measured in a vessel. In addition to 3-Fe ions and complex ligands, the analytical agents contain all the reagents necessary for the particular analysis of a substance, such as enzymes, substrates, co-enzymes, effector substances, antigens, antibodies, etc. These analytic agents may furthermore also contain non-reactive substances, such as, for example, buffers,

  wetting agents and stabilizers. As described above

  above, enzymes which form thiol groups by means of

  of NADH and NADPH as co-enzymes are liponamide-

  dehydrogenase and glutathione reductase. Reagent combinations can be prepared from the enzymes, reagents and substances mentioned, mixed in the form of a solution or a powder or else

  being in the form of tablets or lyophilisate. Lasso-

  Reagent (if not already in the form of a solution) is placed in water or other suitable solvent and a reagent solution is prepared. If the reagent combination consists of separate constituents,

  these must be mixed with each other.

  After mixing the sample (eg, substrate solution, enzyme solution, blood serum, plasma, or urine) with an aliquot of the reagent mixture, the color formed is measured on a photometer and the particular concentration or substrate concentration is calculated using the molecular extinction coefficient and the volumes of reagent or sample added. Kinetic and endpoint measurements are

possible.

  The Fe 3 + / ligand composition mixture may further be fixed by impregnation, together with the reagent or reagents or other enzymes necessary for the detection of a particular parameter, buffer composition, wetting agents and activators when appropriate. and other auxiliary substances on absorbent reagent carriers such as papers, veils, etc. For this purpose, one or more solutions

  impregnating agents can be prepared in the form of

  aqueous, organic or mixed, depending on the

  reagents or substances

  iliary. Absorbent or swellable substrates, preferably filter paper or an absorbent web of glass or plastic, are impregnated or spray-treated with these solutions. The supports are then dehydrated. The reagent carriers thus prepared can be used in the form of rapid diagnostic systems for the direct determination of the contents of a liquid (for example in biological fluids, such as blood, urine or saliva, or in food products, eg fruit juice, milk, etc. The liquid is then directly contacted with the reagent carrier or the reagent carrier is briefly immersed in the liquid A semi-quantitative determination is possible by placing a color reference to the color thus formed A quantitative evaluation may be

  be carried out by photometry-reflectometry.

  It is also possible to introduce the analytical agent according to the present invention into

  matrices as supports, prepared from

  intended to be cast. Examples which may be mentioned herein are cellulose, cellulose derivatives, gelatin, gelatin derivatives or plastics, such as polyurethanes and acrylamide. In this case, it is advantageous to be able to directly add the analytical agent and, where appropriate, the other reagents necessary for the solution to be poured, which means that it is possible to produce the analytical device, consisting of the

  support and reagents, in one operation.

  By eluting the aforementioned reagents

  by means of water, buffer or serum, absorbent

  Alternatively, a reagent solution can be prepared by which it is possible to assay substrates or enzymes in a photometer tank as described above.

  Buffers suitable for the analgesic agents

  mentioned are the phosphate buffers, citrated

  Bates, borates and GOODs containing complementary ions of alkali metals or ammonium. However, other systems can be used as well. The values of

  pH that must be achieved are included in the inter-

  range from 6 to 10, especially from 6.5 to 7.5.

  The wetting agents are, in particular, anionic and cationic wetting agents which undergo ionic interactions with the compounds according to the present invention. However, nonionic wetting agents that activate the enzymes may also

  to be used. Sodium lauryl sulphate, dioctyl sulfonate

  Sodium succinate and alkylarylpolyether alcohols are preferred. The effector substances that can be used are those known for the particular enzymatic reaction. Other auxiliary substances which may be suitable are the usual thickeners, solubilizers, emulsifiers, optical brighteners, contrast media, etc. as known in corresponding tests in association with

other chromogenic substances.

Example

  Detection of thiol groups by means of FeCl and complex ligands

  To detect NADH in the analogue system

  The following constituents serving as reagents were introduced into a tank: Concentration in the analytical medium 1740 of 0.1 M / l acetate buffer pH = 87 mmol / l of 2.5 mmol / liponamide 1 μl of FeCl 3 solution 1 mmol / 1 μl of dipyridyl 3 mmol / l 1 μl of liponamide dehydrogenase 12 μU / l After measurement of the control values of the reagents, the reaction was triggered by the addition of μl of solution of NADH. The maximum extinction measured is 515 nm. Kinetic research has shown a stable end point (extinction variation in 20 minutes = 1%) after an equal reaction time

only at 1 minute.

  To test functional capacity and linearity, NADH concentrations in the range of 1 to 10 ramol / l were measured in the analytical sample. Extinguishing differences

  measured at 515 nm are summarized in Table 1.

Table 1

NADH E51! 5 nm (mmol / l)

1 0.124

0.219

3 0.327

0.429

0.513

6 0.633

7 0.735

0.822

9 0.964

1,098

  The colors that can be obtained with the different complex ligands and the extinction maximums

  correspondents are summarized in Table 2.

1 1

Table 2

  Max Color Formula Max Y H YR rouae 540 2 {"N = N blue 615

N N --- N

3,508

  3N <red H 1 4> CH3 red 510 NOH OH s02 1 purple 556 NH2 OCH v

26 1 0 4 0 9

  Table 2 (continued) Formula Color X N-OH O + f 6 HN-rH blue 650

H3C CH3

  7 Nô red 522 Nô \ 8 purple 552 SO3Na S0> Na tN "9 violet 560

OC8H17

H03S N violet 583

26 104Q09

  Table 2 (continued) Max Color Formula (n) max (nm) CIH3CH3 yellow 441

11 H3 CH3

H2N-N = C --- C = N-NH2

H15C7 C7H15

I I

H2N-N = C ---- C = N-NH2

  It is self-evident that the present invention has been described for explanatory purposes, but in no way limiting and that many modifications can be made without it.

get out of his frame.

Claims (11)

  An analytical agent for the detection of thiol groups, characterized in that it contains Fe3 ions which can be reduced to Fe2 ions by thiol groups and a complex ligand which is capable of forming a   colored complex with Fe2 + ions formed.   2. Analytical agent according to claim 1, characterized in that it is intended for the detection of thiol groups present in the amide of liponic acid,   thiocholine, glutathione and thioglycosides.   3. Analytical agent according to the claims   1 and 2, characterized in that it contains the thiol group in the precursor state in the form of a thioester, a   thio-ether, disulfide or thio-acetal.   4. Analytical agent according to the claims   1 to 3, characterized in that the complex ligand for the 2+ Fe ions is derived from the group comprising ferroines, cuproins and terroines.   5. Analytical agent according to the claims   1 to 4, characterized in that the complex ligand for 2+ Fe ions is derived from the group comprising hydrazones, tetrazolylpyridines, pyridylquinazolines, bis-isoquinolines, imines, phenanthrolines,   bipyridines, terpyridines, bidiazines, pyridyl-   diazines, pyridylbenzimidazoles, diazyltriazines, orthonitroanilines, phenols, tetrazines, quinoxalines, benzimidazoles,   substituted methyl or phenyl-2-pyridyl ketones.   6. Analytical agent according to the claims   1 to 5, characterized in that the complex ligand contains in addition to a hydrophobic radical.   7. Use of the following analytic agent   Claims 1 to 6, characterized in that it is   for the detection of enzymes that cleave compounds thio.   B. Use according to claim 7, characterized in that the enzymes are esterases,   thioglycosidases or thio-esterases.   9. Use of the following analytic agent   Claims 1 to 6, characterized in that it is   intended for the detection of the amide of reduced liponic acid.   10. Use of the following analytic agent   Claims 1 to 6, characterized in that it is   intended for the detection of reduced pyridine nucleotides   by liponamides and liponamide-d: shydrogenase.   11. Enzyme detection method, characterized in that free thiol groups are formed directly or indirectly by the enzymatic reaction and these are detected by means of the analytic agent according to the Claims 1 to 6.   12. Substrate detection method,   characterized in that thiol groups are formed directly or indirectly by the reaction of the substrates and these are detected by means of the following analytic agent Claims 1 to 6.