Tyrosinase mixture

Some non-silica sol-gel materials have also been developed to immobilize bioactive molecules for the construction of biosensors and to synthesize new catalysts for the functional devices. Liu et al. [33] proved that alumina sol-gel was a suitable matrix to improve the immobilization of tyrosinase for detection of trace phenols. Titania is another kind of non-silica material easily obtained from the sol-gel process [34, 35], Luckarift et al. [36] introduced a new method for enzyme immobilization in a bio-mimetic silica support. In this biosilicification process precipitation was catalyzed by the R5 peptide, the repeat unit of the silaffin, which was identified from the diatom Cylindrotheca fusiformis. During the enzyme immobilization in biosilicification the reaction mixture consisted of silicic acid (hydrolyzed tetramethyl orthosilicate) and R5 peptide and enzyme. In the process of precipitation the reaction enzyme was entrapped and nm-sized biosilica-immobilized spheres were formed. Carturan et al. [11] developed a biosil method for the encapsulation of plant and animal cells. 

Selenoureas are prepared by reaction of isoselenocyanates with amines, or by reaction of carbodiimides with a mixture of LiAlH4/Se and by reaction of cyanamides with LiAlH4/Se.267 272 The tyrosinase inhibitory activity and superoxide radical scavenger effect of selenoamides and selenoureas have been investigated (Scheme 84).273 275... 

Waldmann et al. used tyrosinase which is obtained from Agaricus bisporus for the oxidation of phenols to give ortho-quinones via the corresponding catechols in the presence of oxygen (scheme 33).1881 A combination of this enzymatic-initiated domino process with a Diels-Alder reaction yields the functionalized bicyclic components 164 and 165 as a 33 1 mixture starting from simple p-methyl-phenol 160 in the presence of ethyl vinyl ether 163 as an electron rich dienophile via the intermediates 161 and 162 in an overall yield of 77%. 

In fluorine-18 chemistry some enzymatic transformations of compounds already labelled with fluorine-18 have been reported the synthesis of 6-[ F] fluoro-L-DOPA from 4-[ F]catechol by jS-tyrosinase [241], the separation of racemic mixtures of p F]fluoroaromatic amino acids by L-amino acylase [242] and the preparation of the coenzyme uridine diphospho-2-deoxy-2-p F]fluoro-a-o-glucose from [ F]FDG-1-phosphate by UDP-glucose pyrophosphorylase [243]. In living nature compounds exhibiting a carbon-fluorine bond are very rare. 

Two substrates are required in the tyrosinase-catalyzed reaction, phenolic substrate (dopa) and dioxygen. The conditions described in the experiment are such that the reaction mixtures are saturated with dissolved dioxygen. Therefore, when measurements are made for Ku, only the concentration of dopa is limiting, so the rate of the reaction depends on dopa concentration. The dopachrome assay is extremely flexible, as it can be applied to a variety of studies of tyrosinase. 

Whether an inhibitor acts in a competitive or noncompetitive manner is deduced from a Lineweaver-Burk or direct linear plot using varying concentrations of inhibitor and substrate. In separate assays, two substances will be added to the dopa-tyrosinase reaction mixture, and the effect on enzyme activity will be quantified. The structures of the potential inhibitors, cinnamic acid and thiourea, are shown in Figure E5.9. The inhibition assays must be done immediately following the KM studies. To measure inhibition, reaction rates both with and without inhibitor must be used and the tyrosinase activity must not be significantly different. If it is necessary to do the inhibition studies later, the Ku assay for L-dopa must be repeated with freshly prepared tyrosinase solution. 

Muller and co-workers have demonstrated the potential of coupling several spontaneous chemical steps to biocatalysis in a one-pot domino reaction to form bicyclo[2.2.2]octenes.13 A tyrosinase from mushrooms was immobilized on glass beads with the phenol substrate in a mixture of chloroform and a dienophile under air. Tyrosinase can transform a wide variety of phenols to the corresponding catechol, and the presence of air resulted in spontaneous oxidation to the ortho-quinone (Scheme 21.3). The presence of a dienophile then resulted in a Diels-Alder cycloaddition to form the bicyclo[2.2.2]octene product. Significant yields were achieved with a broad range of phenols and dienophiles. 

The standard reaction mixture contained 0.3 fig of mushroom tyrosinase in 300 fiL of 0.05 M Mops buffer (pH 6.5) and 3.6 /tmol of 3,4-dihydroxybenzaldehyde dissolved in 300 fiL of the same buffer. The reaction mixture was incubated at 30°C, and aliquots were withdrawn at various times up to 10 minutes, with each added to an equal volume of ice-cold 0.2 M perchloric acid. After centrifugation, 2 to 5 fiL was analyzed by HPLC. For single time-point determinations, the reaction volume was reduced to 50 fiL. Product formation was linear with time to 10 minutes and with the amount of protein added. 

The following examples will be used to demonstrate that medium-polarity reversed phases have great potential for protein separations. In Fig. 7 the separation of the C apolipoproteins is shown on a phenyl and on a Ci8 column. The Cig column gave almost no separation of the protein mixture, despite the use of a variety of mobile-phase conditions, whereas the phenyl column gave an excellent separation. Lewis et al. (34) found that the columns of intermediate polarity were effective in the separation of tyrosinase, collagen i, cytochrome c, and bovine serum albumin. In this study the diphenyl column was more retentive than the octyl or nitrile... 

Quinones. In the presence of tyrosinase and oxygen, phenols are oxidized to >-quinones which are captured by dienophiles such as ethyl vinyl ether. Chemical oxidation of phenols to p-quinones ° in refluxing benzene (6 examples, 36-65%) is mediated by a mixture of Co and Mn salts of 4-aminobenzoic acid. 

KER, keratin, as detected by a mixture of CAM5.2, MAK-6, and AEl /AE3 EMA, epithelial membrane antigen VIM, vimentin DES, desmin MSA, muscle-specific actin SMA, smooth muscle (alpha isoform) actin CALD, h-caldesmon S-IOOP, S-100 protein TY, tyrosinase Ml, MART-1 (melan-A) OGN, osteocalcin EPS, epithelioid sarcoma EPSS, epithelioid synovial sarcoma EAS, epithelioid angiosarcoma EMPNST, epithelioid malignant peripheral nerve sheath tumor CCS, clear cell sarcoma SEFS, sclerosing epithelioid fibrosarcoma ... 

Krastanov A (2000) Removal of phenols from mixtures by co-immobUized laccase/tyrosinase and Polycar adsorption. J Ind Microbiol Biotechnol 24 383-388 Kuan 1C, Hen M (1993) Stimulation of manganese peroxidase activity a possible role for oxalate in lignin biodegradation. Proc Nad Acad Sci USA 90 1242-1246 Lante A, Crapisi A, Krastanov A et al. (2000) Biodegradation of phenols by laccase immobilised in a membrane reactor. Proc Biochem 36(l-2) 51-58 Lee C, Yoon J, von Gunten U (2007) Oxidative degradation of A-nitrosodimethylamine by conventional ozonation and the advanced oxidation process ozone/hydrogen peroxide. Water Res 41(3) 581-590... 

Monophenol oxidase catalyzes the hydroxylation of monophenols to o-diphenols (Figure 11.1). The enzyme is referred to as tyrosinase in animals, since L-tyrosine is the major monophenolic substrate [37], In mammals, L-tyrosine is the initial substrate in the pathway leading to the final products of black-brown eumelanins, red-yellow pheomelanins, or a mixture of pheomelanins and eumelanins. In plants, the enzyme is sometimes referred to as cresolase owing to the ability of the enzyme to utilize the monophenolic substrate, cresol. In microorganisms and plants, a large number of structurally different monophenols, diphenols, and polyphenols serve as substrates for tyrosinase. As many plants are rich in polyphenols, the name PPO has been frequently used for this enzyme [38]. 

The enzyme/substrate (tyrosinase/wool hydrolysate) mixture (20 pL) was diluted widi 980 pL of distilled water, 70 pL of ethylenediamine, and 50 pL of 2M etfaylenediainine dihydrochloride (pH 11). The mixture was incubated at 50°C for 2 h in die dark, and then the fluorescence intensity was measured using a UVA IS spectrofluorom o (Tecan). The excitation and emission wavelengdis were at 420 and 543 nm, respectively. The concentrations of the DOPA residue in the preparations were estimated using a standard fluorescence curve of DOPA [8]. For the Dopa Quinone (DQ) quantification M6TH (3-methyl-2-benzothiazolinone hydrazone hydrochlcnide monohydrate) was used. MBTH reacts with DQ to form a pink pigmoit widi Xnm at 505 nm. The assay solution was prepared by mixing 480 pL of the en me/substrate reaction mixture, 980 pL of 4.3% (v/v) DMF (dimethyl formamide) in distiUed water, and 580 pL of 20.7 mM MBTH. The total volume was 2 mL. Tbe reacticm mixture was incubated at 25 C for 10 min before the absorbance measurement (at 505 nm) [8]. 

Tyrosinase was dissolved in the reaction mixture before addition of quercetin. The reaction was carried out by adding 0.15 mg of enzyme to 15 mg of quercetin in 100 ml 50% ethanol-buffer. 

Metal salt treatments were conducted in the solution of 50% ethanol Tris buffer (0.5M pH 7.5). A 2 1 molar ratio of mineral salt to quercetin was used in all experiments. The weight ratio of tyrosinase to quercetin was 1 100. The mineral salt and quercetin were dissolved in ethanol-buffer separately before mixing. Tyrosinase was also dissolved separately in a small amount of ethanol-buffer and added to the mineral-quercetin solution. The final volume was then adjusted in a volumetric flask to 10 ml, and the reaction mixture was left standing at room temperature for one hour. The Ames test was then carried out with 0.1 ml portions of the reaction mixture as previously described. 

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