The invention also relates to the use of said substrates for dyeing e.g. keratinous fibers, in particular hair, dyeing textiles, a dyeing composition and a method for dyeing keratinous fibers.
| 20090100610 | Mixture of Sulfide Dyes | April, 2009 | Cremer et al. |
| 20070151042 | Device and method for wet treating laundry | July, 2007 | Bringewatt et al. |
| 20020157190 | Washing apparatus and method of washing laundry | October, 2002 | Imai et al. |
| 20030175226 | Method for gradual hair lightening | September, 2003 | Patel et al. |
| 20070192962 | Aqueous Compositions Containing Alkoxylated Alcohols And Hydrophobic Components, Method For The Production And Use Thereof | August, 2007 | Erhardt et al. |
| 20090165220 | Depilatory products | July, 2009 | Mackles et al. |
| 20100000026 | METHOD FOR WASHING LAUNDRY IN A PROGRAM-CONTROLLED DOMESTIC APPLIANCE, AND CORRESPONDING DOMESTIC APPLIANCE | January, 2010 | Czyzewski et al. |
| 20060021149 | Process for making a leather article structure | February, 2006 | Orietti Carella |
| 20090249560 | LAUNDRY WATER EXTRACTOR SPEED LIMIT CONTROL AND METHOD | October, 2009 | Gaulter et al. |
| 20070079447 | Insect repellent textile | April, 2007 | Kroepke et al. |
| 20070033749 | Anionic azo dyes and the metal complexes thereof ofr colouring leather | February, 2007 | Somogyi et al. |
[0001] The present invention concerns the use of aminobenzoic acid (DABA) as a substitute for e.g. o-phenylendiamine (OPD) in analyses based on peroxidases as well as a dyeing substrate (i.e. a dye precursor) in dyeing compositions, and as an substrate for dyeing natural and synthetic fibres including textiles, thread and yarns. The invention also relates to a composition adapted for dyeing keratinous fibres, e.g. hair, wool, fur and hides, and a method for dyeing such keratinous fibres.
[0002] Immuno-chemical Assays
[0003] Several different substrates are known in the part to be used for enzyme systems in connection with peroxidase-based immuno- hemical assays, for example ELISA. These substrates are often toxic, mutagenic or carcinogenic.
[0004] ELISA (Enzyme Linked Immuno Sorbent Assay) is a method used to assess the amount of antibody in serum. The main principle is that in a much diluted solution many antigens will become bound to the surface of plastic. Thus, if a much diluted solution of antigens is incubated for a time in plastic trays it is possible to wash the cavities in a buffer solution and still retain the film of antigens on the surface of the plastic. If it is required to determine the amount of antibodies in, for instance, serum then the trays with their deposits of antigens are incubated with the serum. The antibodies attach themselves to the antigens and, after thorough washing the trays are again incubated this time with a marker, for example anti-immune globulin serum to which there is attached a suitable enzyme by covalent bonding. In this particular case peroxidase is used.
[0005] The peroxidase-marker complex will attach itself to those locations where there is already a deposit of antibodies. After thorough washing to remove all un-combined material the enzyme activity is measured, normally by the use of a suitable colour indicator. Enzyme activity may be determined by the addition of a cromogenetic substrate (i.e. colour producing compound) and hydrogen peroxide. The enzyme catalyses the reduction of the substrate to a coloured compound and the resultant degree of absorbency provides a measure of the enzyme activity. If the serum contains no antibodies there will be no enzyme activity, on the other hand if there is much antibody present there will be very considerable enzyme activity. A standard curve can be drawn showing enzyme activity as a function of the concentration of antibodies. This may be used to estimate the content of antibodies in unknown serum samples by interpolation.
[0006] Many of the peroxidase substrates are aromatic amines and include diaminobenziden (DAB), 3,3′-diamino benzid tetra hydrochloride, 3,3′,5,5′-tetra methyl benzidin (TMD). Another peroxidase substrate, which does not belong to the group of aromatic amines, is 2,2-azino-di (3-ethyl-benzo thiazolin-6-sulphonic acid) (ABTS), this has been used as a standard for the establishment of the activity of peroxidase preparations. According to Voogd, Van der Stel and Jacobs (1980) this material is also a mutagent.
[0007] o-phenylendiamine (OPD) is another peroxidase substrate which is widely used in hospital and development laboratories. OPD is known to be both mutagenic and carcinogenic.
[0008] The staff of laboratories in which analyses involving the use of toxic, mutagenic or carcinogenic materials are carried out are exposed to a significant degree of risk of coming into direct contact with these materials. In order to provide a safe working environment considerable efforts are now made to substitute these dangerous materials with less dangerous ones.
[0009] Hair Dyeing Composition
[0010] In addition to being used as a substrate in immuno-chemical assays OPD is used to dye hair. In this connection too it is desirable to substitute a dangerous material with one less dangerous so that the user is not exposed to danger by coming into contact with it. To protect the hands against the dangerous material it is normal for gloves to be used while the hair dye is being applied. Gloves cannot, of course, protect the scalp of the person to whom the dye is applied.
[0011] In general hair dyeing compositions on the market today can be divided into three main groups:
[0012] temporary hair dyes,
[0013] semi-permanent hair dyes, and
[0014] permanent oxidative hair dyes.
[0015] The temporary hair dyes are only intended to change the natural hair colour for a short period of time and usually functions by depositing dyes on the surface of the hair. Such hair dyes are easy to remove with normal shampooing.
[0016] When using semi-permanent hair dyes the colour of the dyed hair can survive for five or more shampooings. This is achieved by using dyes having a high affinity for hair keratin and which is able penetrate into the interior of the hair shaft.
[0017] Permanent hair dyes are very durable to sunlight, shampooing and other hair treatments and need only to be refreshed once a month as new hair grows out. With these dyeing systems the dyes are created directly in and on the hair. Small aromatic colourless dye precursors (e.g. p-phenylene-diamine, o-aminophenol, o-phenylendiamine (OPD)) penetrate deep into the hair where said dye precursors are oxidised by an oxidising agent into coloured polymeric compounds. These coloured compounds are larger than the dye precursors and can not be washed out of the hair.
[0018] By including compounds referred to as modifiers (or couplers) in the hair dyeing composition a number of hair colour tints can be obtained. Cathecol and Resorcinol are examples of such modifiers.
[0019] Some of the today most widely used dye precursors such as OPD are known to be both mutagenic and carcinogenic.
[0020] Further, traditionally H
[0021] The use of H
[0022] To overcome the disadvantages of using H
[0023] U.S. Pat. No. 3,251,742 (Revlon) describes a method for dyeing human hair by dye formation in situ (i.e. on the hair). An oxidation enzyme is used for the colour formation reactions at a substantially neutral pH (7-8.5). Laccases, tyrosinases, polyphenolases and catacolases are mentioned as suitable oxidation enzymes. The hair colour pigment is formed by controlled oxidation of various quinone-forming compounds and mono or poly aromatic amines having the amino groups on the aromatic rings to form natural appearing pigments. Specifically mentioned dye precursors are 2-amino-4-nitrophenol, p-phenylene diamine, m-phenylene diamine, o-phenylene diamine, 2-amino-1,4-naphthoquenone, m-aminophenol, p-aminophenol, o-aminophenol, 2-amino resorcinol, 1,2,4-benzene triamine, nitro-p-phenylene diamine, 2-amino-5-diethyl amino toluene.
[0024] EP patent no. 504.005 (Perma S.A.) concerns dyeing compositions for keratinous fibres, in particular hair, which do not require the presence of H
[0025] The aim of the present invention is to use the present findings to make available a substrate which to all intents and purposes is non-toxic, non-mutagenic and/or non-carcinogenic and which may be used in immuno-chemical assays, for the dying of keratinous fibres, in particular hair and for dying both natural and synthetic fibres, e.g. textiles. This aim is achieved by utilising the discovery of a substrate which includes the group with the general formulae shown in 1.
[0026] wherein
[0027] R is an amino, mono- or a distributed amino or OR′, where R is H, alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl, phenyl or substituted phenyl. X, Y and Z may each be any one of the following: alkyl, alkenyl, alkynyl, halogenalkyl, nitro, benzyl, phenyl, substituted phenyl, amino, hydroxy or mercapto with the proviso that at least one of the groups X, Y and Z is an amino group or an amino salt.
[0028] In a special embodiment of the invention a substrate is made available which includes a connection with formula 1 where R′ is a methyl, ethyl or isopropyl group.
[0029] In an preferred embodiment the substrate is a benzoic acid ester, in particular 3,4-diaminobenzoic acid methyl ester (DABA-Me), 3,4-diaminibenzoic acid ethyl ester and 3,4-diamino benzoic acid isopropyl ester.
[0030] Comparison of the very toxic aniline with the carboxyl acid derivative of aniline, p-amino benzoic acid (PABA) shows that the toxicity of the molecule is radically altered by the addition of the carboxyl group. PABA is generally considered to be non-toxic and, among other applications, is used as an ultra violet filter in sun lotions. If the substrate OPD is thought of along the same lines it can be seen that a possible analogue is 3,4-diamino benzoic acid (3,4-DABA). This material is comparatively cheap and readily obtainable.
[0031] Investigation using enzymes showed that 3,4-DABA is a considerably poorer substrate for peroxidase than is OPD. In other words 3,4-DABA has a higher K
[0032] The reaction mechanism for the oxidation of OPD with, for instance, hydrogen peroxide, both with and without an enzyme, is described by the following equations:
[0033] It may be seen from this that the product of oxidation is 2,3-diamino phenazin.
[0034] When one of the compounds described here is employed the product of oxidation is also an amino phenazine. In the case of 3,4-DABA the oxidation product is 4,7-dicarboxy-1,2-diamino phenazine as shown in the following equation.
[0035] The substrate should have two amino groups at the 3,4-location for the reaction to take place. However, substrates which have the amino groups at either the 2,3-location or the 2,3,4-location may be used but only if the carboxyl group does not hinder the reaction.
[0036] Substrates which have the amino group at the para location may also be used to produce a coloured product. An example of this is 3,6-DABA.
[0037] In order to counteract the carboxyl groups inductive effect (i.e. deactivation of the aromatic ring caused by the groups attractive effect upon electrons) esterification of the carboxyl group was investigated using electron donating groups to see if deactivation could be counteracted while at the same time retaining non-mutagenic attributes. To investigate the effect of different alkyl groups on the material's enzymatic properties as well as possible mutagenic properties the methyl, ethyl and isopropyl esters of 3,4-DABA were synthesised.
[0038] Compounds with the general formulae 1 are preferably dissolved in DMF (Dimethyl formamide) but other organic solvents may be used for this purpose. If a compound with the general formulae 1 is in the form of a salt it can be dissolved in water and this is preferred when using organic solvents.
[0039] In another aspect the invention relates to a method for quantitative and/or qualitative analysis of a material of biological interest. In this case a peroxidase enzyme together with a marker is bound to the compound in question. Hydrogen peroxide is then converted with a cromogenetic substrate (i.e. colour forming compound) in the presence of the peroxidase, the substrate includes a bond with the general formulae 1.
[0040] In a preferred embodiment of the method of the invention one of the following substrates are used: the methyl, propyl or isopropyl ester of amino benzoic acid.
[0041] In those cases where the material of biological interest is an antigen the associated antibody is used. In this connection other combinations will suggest themselves to the skilled person.
[0042] The coloured product produced by the method of the invention is especially suited to the dying of textiles, thread, yarn, wool, hides and skins and human hair. Other natural fibres such as cotton and silk may also be dyed with the product as may synthetic fibres such as polyamides, polyurethane and polyester.
[0043] The coloured product may either be made immediately before it is to be used for dying or it may be synthesised in the immediate vicinity of the substance to be dyed. For example this may be done by mixing the substrate and the oxidation system in a person's hair.
[0044] The dying process may be carried out rinsing the person's hair with a mixture of the substrate of the invention and hydrogen peroxide or an oxidation enzyme. A peroxidase is then added and distributed in the hair. When the desired degree of colouring has been obtained the hair is rinsed with water.
[0045] The substrate may be mixed with the oxidation system before it is applied to the hair. As stated above the substrate may be oxidised with hydrogen peroxide or an oxidation enzyme generating hydrogen peroxidase in the presence of a peroxidase.
[0046] Peroxidases belongs to the group of enzymes which is known as the oxidoreductases. The group also includes the classes of enzymes dehydrogenase, oxygenase, oxidase, laccase and related enzymes. These enzymes may also be used for as an oxidation system/agent for e.g. dyeing keratinous fibres, such as hair, wool, fur and hides and the like. Dyeing composition and preferred oxidation enzymes will be described further below.
[0047] In oxidation reactions which are catalysed by the enzyme peroxidase the oxygen donor is hydrogen peroxide which is used as an electron acceptor. Oxidases employ oxygen as an electron acceptor.
[0048] Examples of suitable oxidases include catecholoxidase, laccase and o-amino phenoloxidase.
[0049] Oxidation systems which may be used for the oxidation of the substrate in this connection therefore include peroxidase and hydrogen peroxide as well as oxidases, laccases and related enzymes and oxygen. When the system consists of only an oxidase and oxygen it is only necessary to add the oxidase to the substrate as the oxygen in the air is used as an oxidant.
[0050] Dyeing Composition
[0051] In an aspect the invention relates to a composition in particular adapted for dyeing keratinous fibres comprises, e.g. hair, fur, hide or wool. Comprising 1) at least one oxidation enzyme 2) at least one substrate as defined by the formulae 1 and optionally 3) at least one modifier.
[0052] A preferred use of the composition is as a permanent dye for the dyeing of human hair.
[0053] The oxidation enzyme is as also indicated above an oxidoreductase, i.e. an enzyme classified under the Enzyme Classification number E.C. 1 (Oxidoreductases) in accordance with the Recommendations (1992) of the International Union of Biochemistry and Molecular Biology (IUBMB)) which catalyses oxidoreduction reactions.
[0054] Within the class of oxidoreductase enzymes are preferred enzymes which catalyse the oxidation of a substrate (an electron or hydrogen donor) by acting on oxygen (O
[0055] According to the invention three types of oxidoreductases are specifically contemplated:
[0056] a) Laccases or related enzymes, which act on molecular oxygen and yield water (H
[0057] b) Oxidases, which act on molecular oxygen (O
[0058] c) Peroxidases, which act on peroxide (e.g. H
[0059] Also, enzyme systems which comprise a combination of more than one enzyme from a single class or from different classes among the three types of enzymes are contemplated. In the present specification, although reference will often be made to a single enzyme for the sake of simplicity, it is to be understood that the description is generally applicable to such combinations of more than one enzyme. Further, although the invention is generally described in terms of the preferred aspect relating to the dyeing of hair, it is to be understood that the description is generally applicable to compositions according to the invention adapted for dyeing of other types of keratinous fibres.
[0060] Particularly preferred enzymes are laccases and related enzymes, the term “laccases and related enzymes” including enzymes comprised by the enzyme classification E.C. 1.10.3.2 (laccases) and catechol oxidase enzymes comprised by E.C. 1.10.3.1, bilirubin oxidase enzymes comprised by the enzyme classification E.C. 1.3.3.5 and mono-phenol mono-oxygenase enzymes comprised by the enzyme classification E.C. 1.14.99.1. Laccases are multi-copper containing enzymes that catalyze the oxidation of phenols and aromatic amines. Laccase-mediated oxidation results in the production of aryloxy-radical intermediates from suitable phenolic substrates; the ultimate coupling of the intermediates so produced provides a combination of dimeric, oligomeric, and polymeric reaction products. Certain reaction products can be used to form dyes suitable for dyeing hair.
[0061] Preferably, the laccase employed may be derived from a strain of Polyporus sp., in particular a strain of
[0062] In specific embodiments of the invention the oxidoreductase is a laccase such as a Polyporus sp. laccase, especially the
[0063] Further, the laccase may be a Scytalidium sp. laccase such as the
[0064] The laccase may also be derived from a fungus such as Collybia, Fomes, Lentinus, Pleurotus, Aspergillus, Neurospora, Podospora, Phlebia, e.g.
[0065] Bilirubin oxidase may preferably be derived from a strain of Myrothecium sp., such as
[0066] The substrates (i.e. dye precursors) may according to the dyeing composition of the invention be any of the above within the definition of the general formulae 1.
[0067] Preferred dye precursors (i.e. substrates) are benzoic acid esters, especially diamino benzoic acid esters, in particular 3,4-diamino benzoic acid methyl ester (DABA-Me), 3,4-diamino benzoic acid ethyl ester and 3,4-diamino benzoic acid isopropyl ester.
[0068] Other Oxidation Agents
[0069] The substrate may also be oxidised by a number of inorganic compounds, among these are compounds which include hypochlorite (ClO
[0070] Oxidation systems are taken to be either an oxidant per se or a combination of an enzyme and an oxidant.
[0071] Modifiers
[0072] Modifiers typically incorporation in a dye compositions include m-aromatic diamines, m-aminophenols, polyphenols, amino naphthalines or naphthols. The modifier (coupler) reacts with the dye precursor in the presence of the oxidative enzyme or the like, converting it into a coloured compound. Examples of specific modifiers (couplers) include m-phenylene-diamine, 2,4-diaminoanisole, 1-hydroxynaphthalene (α-naphthol), 1,4-dihydroxybenzene(hydroquinone), 1,5-dihydroxynapthalene, 1,2-dihydroxybenzene(pyrocatechol), 1,3-dihydroxybenzene (resorcinol), 1,3-dihydroxy-2-methylbenzene, 1,3-dihydroxy-4-chlorobenzene(4-chlororesorcinol), 1,2,3,trihydroxybenzene, 1,2,4-trihydroxybenzene, 1,2,4-trihydroxy-5-methylbenzene, and 1,2,4-trihydroxytoluene.
[0073] Method of Dyeing Keratinous Fibres
[0074] In a further aspect the invention relates to a method for dyeing keratinous fibres, in particular hair, fur, hide and wool, using a composition as described above. The dyeing method can be conducted with one or more dye precursors (i.e. substrates of the invention) and optionally in combination with one or more modifiers. The amount of dye precursor(s) and other ingredients used in the composition of the invention for this purpose are in accordance with usual commercial amounts and therefore known for the skilled person. Hair dyeing is typically carried out at or near room temperature, preferably around the optimum temperature of the enzyme being used, and at a pH in the range of from 3.0 to 9.0, preferably 4.0 to 8.5, especially 6.0 to 8.0. Dye precursors (i.e. substrates of the invention) and optional modifiers are described above.
[0075] The invention is further illustrated in the following non-limiting example.
[0076] Synthesis of 3,4-diamino Benzoic Acid Esters
[0077] The esters were made starting from 4-amino-3-nitrotoluene, followed by the esterification and reduction of 4-amino-3-nitrobenzoicacid esters to 3,4-diamino benzoic acidester. The reactions are shown below.
[0078] The amino group in 4-amino-3-nitrotoluene is protected by boiling in anhydrous acetic acid in acetic acid. The methyl group is oxidized with permanganate in an aqueous solution containing magnesium sulphate. The acetyl group is removed by boiling with 0.1 hydrochloric acid. After isolation and drying 4-amino-3-nitrobenzoicacid is dissolved in absolute alcohol and concentrated sulphuric acid is added. On boiling for two to five hours the acid groups are esterified. The exact boiling time depends on the type of alcohol. The last stage is the reduction of the isolated product with activated iron and water in boiling benzine for about five hours.
[0079] When the reaction was complete the iron particles were filtered out and the residue dried for between 24 to 48 hours over anhydrous sodium sulphate. After filtration and evaporation the ester was recrystallised in a mixture of n-butanol and benzine in the ratio of 1 to 10.
[0080] The above procedure was used to produce 3,4-DABA esters for measurement of enzymes and for mutagenetic testing. If larger amounts were to be required at a reasonable price then direct esterification of 3,4-DABA would be preferred. Thus the methyl ester may be prepared by bubbling hydrochloric acid gas through a solution of 3,4-DABA containing methanol. This last method finds only limited use in the production of the ethyl ester and cannot be used to produce the isopropyl ester.
[0081] Characterization of 3,4-DABA Esters
[0082] Thin Layer Chromatography
[0083] The DABA esters that were prepared were analysed and compared with the help of thin layer chromatography (TLC) using silica gel plates of the type MERC 60 FTABLE 1 Chemical bond Rf-value 3,4-DABA 0.15 OPD 0.19 Methyl ester 0.29 Ethyl ester 0.31 Isopropyl ester 0.33
[0084] It can be seen from table 1 that the bigger alkyl groups give a measurably greater displacement in the solvent which contains chloroform. The least displacement is seen, as might be expected, with 3,4-DABA which contains a free carboxyl group. All the combinations tested moved in the form of patches which is an indication of their purity.
[0085] The fact that the 3,4-DABA esters are lipeds gives no problems with regard to solubility in water when making up solutions of substrates. A standard solution of dimethyl formamide diluted in an aqueous buffer with a pH of 5.0. At high concentrations of substrate oxidation products formed by the action of enzymes may cause slight turbidity in the solution. By reducing the pH to about 1 with 1M sulphuric acid a completely clear solution is produced. This is due to the addition of protons to the amino groups.
[0086] Determination of Melting Points
[0087] To verify that the materials synthesised were identical with those described in the literature their melting points were determined and compared with values given in a table on page 1532 of Chapman and Hall's Dictionary of Organic Compounds, Fifth Edition, Volume 2. Melting points were determined by the use of the capillary tube method using a silicone oil bath.
[0088] Melting points are given in Table 2.
TABLE 2 Material Measured melting point Value from literature methyl ester 108-109° C. 108-109° C. ethyl ester 112-113° C. 112-113° C. isopropyl ester 73-74° C. —
[0089] It can be seen from the above table that there is close agreement between the melting points determined by experiment and the melting points as given in the literature. It may, therefore, reasonably be assumed that the synthesised material are identical with those described in the literature. It was not possible to find a value for the isopropyl ester in the literature.
[0090] Ultraviolet Spectroscopy
[0091] UV spectra were taken of OPD as well as the methyl, ethyl and isopropyl esters.
[0092] Scanning was done from 360 nm to 210 nm using a solution of the compounds in methanol. The concentration was 0.1 /1. Table 3 shows the absorption maxima and the extinction coefficients for the compounds investigated.
TABLE 3 Material Absorption max. Extinction coefficient OPD 290.0 nm 2000 M 231.3 nm 3400 M methyl ester 310.0 nm 6000 M 277.5 nm 6000 M 232.5 nm 8400 M ethyl ester 310.0 nm 6200 M 277.5 nm 6000 M 232.5 nm 8600 M isopropyl 310.0 nm 6500 M ester 277.5 nm 6200 M 232.5 nm 8700 M
[0093] As can be seen from the above table all three esters absorb at the same wavelength. At 237.5 nm there is a slight increase in extinction with increasing molecular weight of the alkyl group. 3,4-DABA-esters show a typical maximum at 277.5 nm. This maximum is not found in OPD because of the ester carbonyl group.
[0094] In order to investigate the properties of 3,4-DABA and the three esters with respect to oxidation catalyzed by peroxidase a series of measurements were carried out on the enzymatic reactions initial velocity with increasing concentration of the substrate. Measurements were carried out on OPD,3,4-DABA, 3,4-DABA-methyl ester, 3,4-DABA-ethyl ester, and 3,4-DABA isopropyl ester.
[0095] Absorbancy at 492 nm was used as a measure of the course of the reaction. The initial velocity was taken to be the absorbancy at 492 nm two minutes after the addition of the peroxidase to a mixture of the substrate and hydrogen peroxide in a buffer with a pH of 5.0. The wavelength of 492 nm was chosen because it is employed in the standard assay procedures for OPD. None of the substrates has a specially high absorption at this wavelength.
[0096] By varying the concentration of the substrate and at the same time measuring the initial velocity of the reaction it is posible to apply the Michaelis/Menten equation to the system.
[0097] Under ideal conditions the values determined experimentally will approach the value of the expression:
[0098] where [S] is the concentration of the substrate, V
[0099] The expression implies that the initial velocity will increase with increasing concentration of the substrate, but the curve for the velocity will flatten out and approach V
[0100] The peroxidase system has an extremely complicated energy balance (Arnoa et al. (1990)) for short periods of less than a minute the system may nevertheless be described in terms of the above given formulae.
[0101] Enzymatic Determination
[0102] The following solutions were prepared for use in the enzyme assay:
[0103] a) Assay buffer
[0104] A 50 mM phosphate/citrate buffer with a pH of 5.0 was made by mixing a 50 mm Na
[0105] b) DMF diluted 1:10
[0106] By means of pipette 10 ml were placed in a graduated flask which was then topped up to 100 ml with assay buffer.
[0107] c) Solution of hydrogen peroxide 0.018%
[0108] 15 μl of a 30% solution of hydrogen peroxide (PERHYDROL, Merck) was thinned down with 25 ml of the assay buffer.
[0109] d) Stabilizing buffer for peroxidase
[0110] The stabilizing buffer for peroxidase, i.e. a buffer which stabilizes the enzyme, was prepared according to the method of Olsen and Little (1983). A 0.1M Na-acetate buffer, which was 0.5M in terms of CaCl
[0111] 37.5 mg N-acetyl-trimethyl-ammonium-bromide was dissolved in 75 ml of the buffer. To this solution 25 ml of glycerol was added. The enzyme activity is maintained in this buffer because the molecules of the enzyme are prevented from aggregating.
[0112] e) Standard solution of peroxidase
[0113] 10 mg of horseradish peroxidase type VI-A (Sigma no. 6782) were dissolved in 10 ml of the stabilizing buffer. This was stored at −15° C. This will keep for several months (Olsen and Little (1983).
[0114] f) Bench solution of peroxidase
[0115] The standard peroxidase solution was diluted to 1:1000 with the 10 ml assay-buffer solution in 10 μl standard solution.
[0116] g) Standard solutions of the substrates
[0117] 0.5 mmol of each substrate in 5 ml DMF. These solutions will keep for several weeks at −15° C.
[0118] h) Bench solutions of the substrates
[0119] The standard solutions were diluted by 1:10 with the assay buffer. Before being used the substrate solutions were diluted by 1:10. 0.5 ml of the standard solution was thinned down with 4.5 ml of the assay buffer.
[0120] Measurement of the Velocity of Reaction as a Function of the Concentration of the Substrates
[0121] For each of the compounds OPD, 3,4-DABA-, and the methyl, ethyl, and isopropyl esters of 3,4-DABA 15 measurements were carried out and absorbency was measured twice in each case at 492 nm one minute after the addition of 100 μl dilute peroxidase solution (bench solution). The concentration of the peroxidase was held constant at 50 ng/ml during the whole investigation. By using the volume of substrate and the volumes of 10 vol-% DMF solution as given in table 4 it was possible to employ a constant reaction volume and a constant concentration of DMF. For all measurements there was used 1550 μl buffer and 50 μl bench solution of peroxidase. The total volume is then as follows: 300 μI DMF and substrate solution, 1550 μl buffer, 100 μl bench solution of peroxidase and 50 μl hydrogen peroxide solution. That is 2000 μl in total.
TABLE 4 Substrate concentration solution solution ( 0 300 0 5 295 25 10 290 50 15 285 75 20 280 100 25 275 125 30 270 150 35 265 175 40 260 200 50 250 250 60 240 300 80 220 400 100 200 500 150 150 750 200 100 1000 300 0 1500
[0122] Table 5 contains information about the values measured for KTABLE 5 K V K Substrate ( (min (1*mol OPD 47.35 0.16 1.6 * 10 3,4-DABA 251.22 0.20 2.0 * 10 methyl 125.97 0.22 2.2 * 10 ester ethyl 212.39 0.27 2.7 * 10 ester isopropyl 118.46 0.22 2.2 * 10 ester
[0123] The results of the measurements of initial velocities are stated in units of absorbency and not in molar units. In order to be able to measure the “true” velocity of reaction it is necessary to isolate the oxidation product for each substrate and determine the molar extinction coefficient. The value of Kkat is worked out from 1 mole of peroxidase of 50,000 g/mol.
[0124] FIG.
[0125] As is shown by Table 5 and FIGS. 1 to 6 the carboxyl esters of 3,4-DABA are effective substrates in a peroxidase/hydrogen peroxide system. It is possible to obtain much higher initial velocities with these compounds than with OPD. K
[0126] All reaction velocities are expressed as absorption units, this is partly because most practical enzyme essays are based on the measurement of absorption and partly because the products of reaction are not isolated from the reaction mixture.
[0127] Determination of the Mutagenicity of the Compounds
[0128] The Ames test (Maron and Ames (1983)) was used to determine the mutagenic properties of the compounds. Nutrient media were prepared in the manner described by Venitt and Parry (1984). To 3×2 ml melted agar at 45° C. were added 100 μl of 50, 100 and 200 mM of solutions of the compounds dissolved in DMSO. By means of a pipette 100 μl of a well-grown culture of
[0129] OPD is not a direct mutagen, it must first be activated by the liver enzyme system P450. All trials were therefore carried out both with and without the addition of “S9-mix” from the livers of rats, this contains the P450 system. 0.5 ml of “59-mix” was added to each test-tube. The criteria for mutation are that increasing concentrations of the compound under investigation give a marked increase in the number of His+ mutants. The results of these trial are given in FIG.
[0130] Dyeing Effect of Dye Precursors of the Invention
[0131] The permanent oxidative dyeing effect of different dye precursors using 0.05 mg active enzyme protein
[0132] The dye precursors tested were
[0133] 0.1% w/w 3,4 diamino benzoic acid (DABA) in 0.1M K-phosphatebuffer, pH 7.0.
[0134]
[0135] Modifier used
[0136] 0.1% w/w m-phenylenediamine (MPD) in 0.1M K-phosphatebuffer, pH 7.0.
[0137] Dye precursor solutions were prepared by mixing the indicated modifier so that the final concentration in the dyeing solution was 0.1% w/w with respect to dye precursor (i.e. substrate of the invention) and 0.1% w/w with respect to modifier.
[0138] Hair Dyeing
[0139] 1 gram 6″ De Meo Virgin natural white hair tresses (De Meo Brothers Inc. USA) were used.
[0140] 4 ml dye precursor solution (including modifier) was mixed with 1 ml laccase on a Whirley mixer, applied to the hair tresses and incubated at 30° C. for 30 minutes.
[0141] The hair tresses were then rinsed with running water, washed with shampoo, rinsed with water, combed, and air dried.
[0142] a*, b* and L* were determined on the Chroma Meter and ΔE* was then calculated as described below.
[0143] Hair tress samples treated without enzyme were used as a blind.
[0144] The result of the test is shown in Table 6.
TABLE 6 DABA and DABA-Me 0.1% w/w dye with/without MPD precursor/modifier Δ Δ Δ Δ Assessment DABA −4.27 −0.63 −2.55 5.01 no colour DABA + MPD −23 −2.21 −18.29 29.47 grayish DABA-Me −7.58 6.53 −2.14 10.23 light orange DABA-Me + MPD −32.31 2.35 −25.07 40.96 grayish violet
[0145] Assessment of the Hair Colour
[0146] The quantitative colour of the hair tresses was determined on a Minolta CR200 Chroma Meter by the use the parameters L* (“0”=black and “100”=white), a* (“−”=green and “+”=red) and b* (“−” blue and “+” yellow).
[0147] ΔL*, Δa* and Δb* are the delta values of L*, a* and b* respectively compared to L*, a* and b* of untreated hair (e.g. ΔL*=L*
[0148] ΔE* was calculated as ΔE*=square root(ΔL*
[0149] Dyeing Effect of DAB-Me and Various Modifiers
[0150] Using the procedure described in Example 4 the permanent dyeing effect of the dye precursor (i.e. substrates) DABA-Me with various modifiers were tested, except that 0.2% w/w dye precursor and 0.2% modifier were used.
TABLE 2 0.2% DABA-Me and 0.2% w/w modifier Δ Δ Δ Δ Assessment 4-chlor-resorcinol −22.36 1.19 −6.28 23.26 Gray-green 5-amino-o-cresol −14.1 5.69 −2.1 15.35 light orange m-phenylene-diamine −33.25 2.17 −23.71 40.9 Grey (Bluish) pyrogallol −29.47 5.74 −7.69 30.99 Brown 4-methoxy-1,3-phenyl- −39.24 2.33 −19.73 43.98 Brown-gray/ enediamine black
[0151] As can be seen the keratinous fibers can be dyed using DABA-Me and a modifier.