2,3-DIHYDROXYBENZOIC ACID AS A SUBSTRATE FOR MUSHROOM TYROSINASE1,2

When 2, 3-dihydroxybenzoic acid (2, 3-DBA) is acted upon by mushroom tyrosinase, a yellow intermediate, 2, 3-DBA-o-quinone, characterized by a peak at 415 nm, is the first product detected. 2, 3-DBA-o-quinone gives rise to a “final blue product” (λmax = 230, 410, 620 nm), and to “soluble oxidation product(s)” (λmax = 275–280, 350–360 nm). Kinetic data (assayed spectrophotometrically and polarographically) obtained when different concentrations of 2, 3-DBA were oxidized by a fixed amount of mushroom tyrosinase, deviated from classic Michaelis-Menten kinetics. Reduction of the “final blue product” with ascorbate resulted in the loss of the blue chromophore at 620 nm and the concomitant appearance of a “yellowish reduced final product.” The “yellowish reduced final product” could be reoxidized with either mushroom tyrosinase or with NaIO4 to the “final blue product,” indicating that the latter has carbonylic quinonoid groups in ortho position to each other.     

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.     

Effect of Proteins, Protein Hydrolyzates and Amino Acids on o-Dihydroxyphenolase Activity of Polyphenol Oxidase of Mushroom, Avocado, and Banana

Casein hydrolyzate and bovine serum albumin did not inhibit the o-dihydroxyphenolase activity of polyphenol oxidase of avocado and mushroom. L-lysine, glycine, L-histidine and L-phenylalanine, in increasing order of effectiveness, inhibited o-dihydroxyphenolase activity to a maximum of about 60% inhibition only; 50% inhibition was observed with 50 mM L-phenylalanine vs. 160 mM L-lysine. L-cysteine at about 0.4 mM gave full inhibition. Triglycine, diglytine and glycine, in decreasing order, were effective in lowering the final level of colored melanin formed by the action of polyphenol oxidase on DL-DOPA. Amino acids are nontoxic and could be safely added to food during processins as a means of preventing undesirable enzymatic browning.     

EFFECT OF MALTOL ON THE OXIDATION OF o-DIHYDROXYPHENOLS BY MUSHROOM TYROSINASE AND BY SODIUM PERIODATE1

Maltol (3-hydroxy-2-methyl-4H-pyran-4-one) inhibits the rate of oxidation of different o-dihydroxyphenols by tyrosinase when assayed spectrophotometrically, but not when assayed polarographically. The spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase (or by sodium periodate) in the absence or presence of maltol were different, suggesting that maltol conjugates with the o-quinones formed. Maltol does not inhibit tyrosinase activity per se but only gives an apparent inhibition probably due to its ability to conjugate with o-quinones.     

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.     

EFFECT OF ACETOHYDROXAMIC ACID (AHA) AND SALICYLHYDROXAMIC ACID (SHAM) ON THE OXIDATION OF o-DIHYDROXY- AND TRIHYDROXYPHENOLS BY MUSHROOM TYROSINASE

Acetohydroxamic acid (AHA) and salicylhydroxamic acid (SHAM) each inhibited the rate of oxidation of different o-dihydroxy- and trihydroxyphenols by tyrosinase when assayed spectrophotometrically or polarographically. SHAM was a much more effective inhibitor than AHA. Spectral changes occurring during the oxidation of different o-dihydroxyphenols by tyrosinase in the presence of AHA or SHAM were different than the spectral changes occurring in their absence. AHA and SHAM also had an effect on the spectrum of the final product(s) formed when different o-dihydroxyphenols were oxidized by the enzyme, suggesting that AHA and SHAM conjugate with the o-quinones formed. A lack of an effect of AHA and SHAM on the spectrum of product(s) formed when trihydroxyphenols were oxidized by tyrosinase suggest that AHA and SHAM do not conjugate with the o-quinones derived from trihydroxyphenols.     

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.