EFFECT OF SALICYLHYDROXAMIC ACID (SHAM) ON DL-DOPA OXIDATION BY MUSHROOM TYROSINASE AND BY NaIO4

Salicylhydroxamic acid (SHAM) inhibits very effectively the rate of DL‐DOPA oxidation by mushroom tyrosinase. SHAM also affects the spectrum of the initial produces) formed when DL‐DOPA is oxidized by mushroom tyrosinase or by NaIO4. Moreover, at certain concentrations, SHAM prevents the polymerization of dopaquinone formed enzymaticatty or nonenzymatically probably due to a chemical interaction between dopaquinone and SHAM.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid [5-hydroxy-2-(hydroxymethyl)-4-pyrone] inhibited effectively the rate of pigment formation during the oxidation of pyrogallol, 2, 3,4-THAP (2, 3,4-trihydroxyacetophenone) and 2, 4,5-THBP (2, 4,5-trihydroxybutyrophenone) by tyrosinase. On the other hand, kojic acid had a synergistic effect on the rate of methyl gallate and n-propyl gallate oxidation to pigmented product(s) (λ_max= 360 nm and λ_max= 380 nm, respectively). However, kojic acid inhibited effectively the rate of oxygen uptake when each of the above trihydroxyphenols was oxidized by tyrosinase. These results suggest that kojic acid inhibits tyrosinase per se (probably due to its ability to bind copper at the active site of the enzyme) and that it exerts only an apparent stimulatory effect during the formation of pigmented product (s) from methyl gallate and n-propyl gallate. Proof for the latter was obtained by a time-course experiment of kojic acid addition and examination of the spectra of pigmented product(s) formed in the absence versus presence of kojic acid, which suggested that the o-quinone of n-propyl gallate and the o-quinone of methyl gallate can each convert kojic acid to a yellow product(s) absorbing at the 360–380 nm region.     

p-HYDROXYPHENYLPROPIONIC ACID (PHPPA) AND 3,4-DIHYDROXYPHENYLPROPIONIC ACID (3,4-DPPA) AS SUBSTRATES FOR MUSHROOM TYROSINASE

p-Hydroxyphenylpropionic acid (PHPPA) and 3,4- dihydroxyphenylpropionic acid (3,4-DPPA) serve as substrates for tyrosinase. The Km value of 3,4-DPPA for tyrosinase is 1.3 mM. The yellow o-quinone of 3,4-DPPA (4-carboxyethyl-o-benzoquinone) (λ_max= 400nm), is detected initially and it is then converted to a red product(s) (λ_max= 480±10 nm), the o-quinone of 6,7-dihydroxy 3-dihydrocumarin (dihydroesculetin). When the concentration of the latter is relatively high, it polymerizes to a final brown product(s), characterized by an ill-defined spectrum.
H₂O₂ shortens the lag period of PHPPA hydroxylation, hastens the conversion of the yellow o-quinone of 3,4-DPPA to the red o-quinone of dihydroesculetin, and prevents the polymerization of the latter to the final brown product(s).
The relatively unstable o-quinone of 3,4-DPPA interacts with amines such as hydroxylamine (NH₂OH), p-aminosalicylic acid (PASA) and p-aminobenzoic acid (PABA), forming relatively stable final product(s) characterized by different spectra from those formed in their absence.
Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each has an effect on the spectrum of product(s) obtained when 3,4-DPPA is oxidized by tyrosinase, indicating that these hydroxamic acids derivatives interact with the o-quinone of 3,4-DPPA. The spectrum of the final product(s) was also different when 3,4-DPPA was oxidized by tyrosinase in the presence of benzenesulfinic acid than in its absence, suggesting the formation of a stable phenylsulfonyl derivative.     

KOJIC ACID CONVERSION TO A YELLOW PRODUCT(S) VIA THE HORSERADISH PEROXIDASE/H2O2 SYSTEM1

Horseradish peroxidase in the presence of hydrogen peroxide oxidizes kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) to a yellow product(s). The yellow product(s) formed has a major absorbance peak at 375 nm and is fluorescent. The relationships between, and effects of, various concentrations of horseradish peroxidase, kojic acid and hydrogen peroxide on the rate of oxidation of kojic acid to the yellow product(s) are described. The observation that the oxidation of kojic acid to the yellow product(s) occurs best in the presence of very low concentrations of hydrogen peroxide, relative to that of kojic acid, suggests that kojic acid is a poor hydrogen donor (AH₂) for horseradish peroxidase.     

EFFECT OF BENZOIC ACID AND SOME OF ITS DERIVATIVES ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

Vanillic acid and salicylic acid inhibited the rate of dihydroxyphenylalanine (DL-DOPA) oxidation to dopachrome (γ_max=475 nm) by tyrosinase at all concentrations tested. Benzoic acid and p-hydroxybenzoic acid (PHBA), at relatively low concentrations, slightly stimulated the rate of DL-DOPA oxidation, whereas at higher concentrations each inhibited the reaction. p-Hydroxybenzoic acid methyl ester (PHBAME), at relatively low concentrations, had a pronounced synergistic effect on the reaction, whereas at relatively high concentrations it inhibited the rate of DL-DOPA oxidation. The synergistic effect of 1.6–6.6 mM PHBAME on the rate of DL-DOPA oxidation to dopachrome was found to be only an apparent effect due to the ability of PHBAME to be hydroxylated very slowly by tyrosinase to a yellow pigmented product(s) with DL-DOPA serving as a reductant (AH₂) for the hydroxylation reaction, thus hastening the conversion of PHBAME to pigmented product(s). Vanillic acid, salicylic acid, benzoic acid and PHBA could not be hydroxylated by tyrosinase.     

EFFECT OF 2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AND 2,4,5-TRIHYDROXYBUTYROPHENONE (2,4,5-THBP) ON THE RATE OF DL-DOPA OXIDATION BY MUSHROOM TYROSINASE1

Various concentrations of 2,3,4-trihydroxyacetophenone (2,3,4-THAP) and 2,4,5-trihydroxybutyrophenone (2,4,5-THBP) have a synergistic effect on the rate of DL-3,4-dihydroxyphenylalanine (DL-DOPA) oxidation by mushroom tyrosinase as measured by absorbance at 475 nm. The synergism results from the ability of dopaquinone to nonenzymatically oxidize 2,3,4-THAP and 2,4,5-THBP to the corresponding o-quinone. Moreover, various concentrations of 2,3,4-THAP and 2,4,5-THBP prevent the enzymatic conversion of DL-DOPA to DOPA-melanin, probably due to the ability of these trihydroxyphenols to interact nonenzymatically with dopaquinone.     

EFFECT OF KOJIC ACID ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE1

Kojic acid is a very effective inhibitor of mushroom tyrosinase as judged by its effect on the rate of pigmented products formation and on the rate of oxygen uptake when different o-dihydroxyphenols are oxidized by the enzyme. In addition to the ability of kojic acid to inhibit the enzyme per se, the data show that kojic acid can change the spectrum of some pigmented products formed in its absence, probably due to the ability of some o-quinones formed enzymatically to oxidize kojic acid to a yellow product(s). These findings call for caution in the use of kojic acid as an inhibitor of enzymatic browning in tissues and processed foods that are not yellow originally.     

2,4,5-TRIHYDROXYBUTYROPHENONE (2,4,5-THBP) AS A SUBSTRATE FOR HORSERADISH PEROXIDASE1

2,4,5-trihydroxybutyrophenone (2,4,5-THBP), in the presence of hydrogen peroxide (H₂O₂), is a substrate for horseradish peroxidase (HRP) with a Km value of 2.5 mM. An intermediate red product(s), probably 2,4,5-THBP quinone, characterized by a peak at 490–500 nm, was detected and the time course of its conversion to the final red product(s) was studied. The relationships between the rate of 2,4,5-THBP oxidation to pigmented product(s) as a function of various concentrations of H₂O₂, 2,4,5-THBP and HPR are described.     

EFFECT OF HYDROXYLAMINE, p-AMINOBENZOIC ACID AND p-AMINOSALICYLIC ACID ON THE OXIDATION OF O-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

The effect of hydroxylamine (NH₂OH), p-aminobenzoic acid (PABA) and p-aminosalicylic acid (PASA) on the spectrum of the final product (s) formed when o-dihydroxy- and trihydroxyphenols were oxidized by tyrosinase was examined. New pigmented product(s), probably oximes, were formed by the interaction of NH₂OH with the o-quinones of 4-methyl catechol, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylpropionic acid (3,4-DPPA) but not with the o-quinones of catechol or protocatechuic acid. Interaction of PABA or PASA with the o-quinones of catechol, 4-methyl catechol, protocatechuic acid, DOPAC and 3,4-DPPA also yielded pigmented oximes. The interaction of the o-quinones of trihydroxyphenols with NH₂OH, PABA or PASA had little effect on the spectrum of the final product (s), suggesting that oximes are not formed in these reactions.     

EFFECT OF BENZENESULFINIC ACID ON THE OXIDATION OF o-DIHYDROXY - AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Benzenesulfinic acid inhibits the rate of oxidation of different o-dihydroxy-phenols and trihydroxyphenols by tyrosinase when assayed spectrophotometrical-ly but barely has an effect when assayed polarographically. Benzenesulfinic acid is a much more effective inhibitor of the rate of oxidation to pigmented product(s) by tyrosinase of o-dihydroxyphenols than of trihydroxyphenols. The spectrum of the final product(s) formed by the oxidation of most of the tested substrates was different in the absence versus presence of benzenesulfinic acid suggesting that the latter traps the o-quinones forming phenylsulfonyl derivatives in the reaction.