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.     

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 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 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 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.     

Some Biochemical Properties of Soluble and Bound Potato Tuber Peroxidase

Soluble and bound forms of peroxidase were extracted from potato tubers using 0.05M sodium phosphate buffer (pH 6.0) and the same buffer containing 0.811 KC1 (pH 6.0), respectively. The soluble and bound potato tuber peroxidase (PTP) fractions represented about 60 and 40%, respectively, of the total peroxidase extracted. Soluble PTP consisted of high levels of protein and low peroxidase activity relative to bound PTP, with the specific activity of the former being about 15-fold lower than that of the latter. The biochemical properties of soluble and bound PTP in both the crude and the partially purified form were studied. The pH curves of soluble PTP and bound PTP were similar with a broad pH optima around 5.0–6.0. The stability of both forms of peroxidases to heat was also similar, with about 50% activity being lost after heating ffor 5 min at 70°C. The isoenzyme profile of equal amounts of activity of soluble PTP and bound PTP was different; anodic gel electrophoresis yielded ten and three isoenzymes, respectively, while cathodic runs showed the same number of isoenzymes in either fraction but with the isoenzymes of bound PTP being detected faster than those of soluble PTP. The contents of proteins and carbohydrates were much higher in the soluble PTP than in the bound PTP fraction. Partial purification of either forms of the enzyme was achieved by column chromatography on Sephadex G-75 or on Sepharose 6B. Chromatography on Sepharose 6B resolved soluble PTP into one major peak (II) and one minor peak (I). Under identical conditions, bound PTP was resolved into two peaks with 80% and 20% of the activity in peaks II and III, jespectively. Column chromatography did not aid in resolving the isoperoxides. The molecular weight of peak II of soluble PTP and of bound PTP was estimated to be about 45,000, while that of peak III of bound PTP was 30,000. From analysis of the 30—90% ammonium sulfate fraction or of the partially purified enzyme on Concanavalin A-Sepharose, it was concluded that the isoenzymes of soluble PTP and of bound PTP differ in their carbohydrate contem and/or composition or in the structure of their carbohydrate units.     

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.