Humic acids-tyrosinase interactions as a model of soil humic-enzyme complexes

Soil Bid. Biochem.Vol. 20, No. 3, pp. 353-359, 1988

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Copyright Q 1988 Pcrgamon Press pk

HUMIC ACID~~ROSINASE INTE~CTIONS AS A MODEL OF SOIL HUMIC-ENZYME COMPLEXES P. RUCZXERO and V. M. RALXX~NA Istituto di Chimica Agraria, Univenita’ degli Studi, Via G. Amendola 165/a, 70126 Bari, Italy (Accepted 20 October 1987) Summary-Interactions between tyrosinase and humic acids (HA), which are of great importance for soil biochemistry, have been studied with respect to binding mechanisms involved and to alterations of the activity and of some properties of the enzyme. Neutralized solutions of soii HA slightly inhibited tyrosinase activity. The extent of inhibition (up to 14.0%) was related to HA ~ncentration. Stable and insotuble HA-tyrosinase complexes were obtained by cofloccufation of HA and enzyme with Ca2+. Depending on the HA concentration and probably on the pH, from 32 to 76% of the added enzyme was strongly bound. The data from i.r. spectroscopy suggest the involvement of carboxyl and phenolic groups of HA in ionic and hydrogen bond formation with the enzyme molecule. Some properties of the HA-tyrosinase complexes and the soluble enzyme were also compared. When tyrosinase was bound by HA, it showed a substrate specificity similar to that of the free enzyme but a K,,, value slightly higher and a thermal stability and resistance to proteolysis lower than those observed for the free enzyme. Although the systems studied were artificial, they are helpful in unders~nding the nature of humic-enqme complexes which apparently occur in the soil.

IMRODUCIION

In soil, extracellular enzymes function in an environment where both enzyme and substrate are often adsorbed or bound to clays and, in particular, humic colloids. The relationships of the enzymes to substrates and microorganisms within their immediate environment have been discussed (Bums, 1982; Stotzky and Bums, 1982) but although many of the physical and chemical properties of the potential interactants are known, questions remain concerning the chemical nature of the humic acids (HA)-enzyme interactions and the consequences of these associations for the microbial ecology of soil. A useful approach toward understanding the complex relationship between soil enzymes and their poiyanionic supports is to prepare model HA-enzyme copolymers. For this purpose, extracted soil HA can be used (Maignan, 1982, 1983; Sarkar, 1986) or model phenoiics can be constructed from the structural units of humic substances (Rowe11 er al., 1973; Sarkar and Bums, 1984). The activity of the enzyme is usually affected by binding to the natural or synthetic HA. The effects of HA on enzyme activities and the properties of synthetic humic-enzyme complexes must be investigated in order to reveal if these analogues truly reflect the state of enzymes in soil. Tyrosinase is a copper-containing enzyme, a poly phenoloxidase, which catalyzes the oxidation with molecular oxygen of several phenolic compounds of plant and microbial origin. This oxidation is believed to be a key step in the synthesis of soil humic matter (Stevenson, 1982). Substrate transformations, catalyzed by polyphenoloxidases, include degradation, polymerization, synthesis, coupling reactions and incorporation into humic substances. These reactions are not limited to naturally-suing phenolic com353

pounds, but include phenols and aromatic amines which are inte~ediates of indust~al wastes and the basic structural units of phenolic and anilinic pesticides (Sjoblad and Bollag, 1981; Bollag, 1983). We investigated the formation of complexes tetween soil HA and mushroom tyrosinase to obtain information on the effects of HA on the enzyme activity, the quantity of enzyme retained in complexes, the nature of the binding m~hanisms involved in the interaction processes. and the extent of any changes occurring in the properties of enzymatic component after complex formation. MATERIALS AND METHODS ~~rer~~~s

Tyrosinase (EC 1.14.18.1 monophenolmonooxygenase) from mushroom (Sigma Chemical Co.) was used without further purification. HA was obtained from the A, horizon of an Andosol in Mount Vulture (Italy) by extraction with 0.5 M NaOH under N,. HA was precipitated by bringing the alkaline extract to pH 2.0 with HCI. HA was then purified by dialysis against water acidified at pH 2.0 and then against distilled water (dialysis tubing Spectrapor 6, Spectrum Medical Ind., cutoff 8000 Da), by passage over OH- and H-saturated exchange resins, and by treatment with HCI-HF solution (Ruggiero et al., 1980). yielding a product of ash content 1.75%. inpUence of the HA on enzyme activity The effect of HA on enzyme activity was assessed in 50mM phosphate buffer pH7.0 by measuring the enzyme-catalyzed reaction in the presence of increasing concentrations of HA after incubation of the samples for 6 h at 25°C. Controls contained the enzyme without HA or the HA alone.

354

Separation of the humic-en:yme

P. RUXERO and

complex

To the same solutions and controls prepared for studying the effect of HA on tyrosinase activity, were added 2 ml of CaCl,, 0.5 M. FIocculation of HA and tyrosinase and a decrease of pH to 4.5 were observed. The HA-enzyme mixture was shaken gently for 6 h at 2O’C. The suspension was centrifuged at 20,OOQg and the pellet washed five times with 0.1 M calcium acetate buffer pH 4.5 until the washings did not show tyrosinase activity. The supematant and the pooled washings of each sample were dialyzed against Na phosphate buffer pH 7.0 and concentrated using polyethylene glycol as the dehydrating agent (Aquatide III, Calbiochem). All the solutions obtained and the insoluble pellet (resuspended in acetate buffer pH4.5) were separately assayed for tyrosinase activity. infrared spectra The HA preparations and HA-tyrosinase complexes were dialyzed against distilled water (dialysis tubing Spectrapor 6, Spectrum Medical Ind., cutoff 2000 Da), concentrated by Aquacide III and then lyophilyzed. The materials obtained and the original tyrosinase were dried in a vacuum desiccator over P,O, and run as KBr pellets (l-2 mg per 400 mg of KBr) on a Pcrkin Elmer model 399B IR spectrophotometer.

v.

M. RaoGs.4

Isoelectric focusing of tyrosinase The isoelectric point of commercially-purified tyrosinase was dete~n~ by analytical isoelectric focusing in thin-layer poiyacrilamide get (Ampholine PAG-plates. LKB, Sweden) within the pH range 3.5-9.5, following the procedure described by LKB (Righetti, 1983). The enzyme was located by staining with Coomassie blue R-250 for protein and with catechol for activity (Galeazzi and Sgarbieri, 1981) after the pH gradient was determined.

Tyrosinase activity was assayed by a polarographic method, using catechol as the substrate. O2 consumption during the enzymatic reaction was measured at 37’C with a Clark-type oxygen electrode (Mayaudon et al.. 1973; Ruggiero and Radogna, 1984). The total volume of solution or suspension was adjusted to 3.0 ml in all experiments. The concentration of catechol in the reaction mixture was 6 m&i!. Unless otherwise specified, phosphate buffer, pH 7.0, was used in all assays. One unit of activity was defined as the amount of catalyst which caused the uptake of I idrnmolof 0, min-I.

RESULTS Isoelectric focusing of tyrosinase

Tyrosinase solutions and HA-tyrosinase suspensions were used for the determination of Michaelis constants. The same volume (0.1 ml) was always injected into the reaction vessel. The kinetic analysis was carried out in 0.1 M calcium acetate buffer pH 4.5. Final concentrations of catechol in the reaction mixture ranged from 0. I to 10 mM. K,,, values were determined by analysis of Lineweaver-Burk. Eadie-Hofstee and Hanes-Woolf plots. Best-fit lines and correlation coeffcients were derived from computer least-square analyses. Stability studies For the study of the enzyme stability toward proteolytic enzymes, a bacterial protease (Type IV, Sigma) was used. Tyrosinase solutions and humic-enzyme suspensions were allowed to react with protease (1 mg ml- ’ ) at 25’C in a shaking water bath for 24 h. Parallel experiments without protease were performed. The mixtures were then assayed for tyrosinase activity, To study enzyme thermostability, the tyrosinase activity of the free and complexed enzyme, before and after 2 h incubation at 37.45 and 55°C was measured.

Analytical el~trofocusing in a thin-layer polyacrilamide gel showed that commercially-purified mushroom tyrosinase was heterogeneous both for protein and for activity. The most likely isoelectric point for the enzyme was at pH 5.0. influence of the HA on enzyme acticity The effect of HA on tyrosinase activity is shown in Fig. 1. With HA: tyrosinase ratios ranging from 0.25 to 5.0 (w/w), the HA was found to reduce tyrosinase activity from between 3.8 and 14.0%. The inhibitory effects on tyrosinase activity produced by HA were constant over 6 h prior to incubation. The blanks containing the same concentration of HA but without enzyme showed no activity.

Substrate specificity For activity assays, some di- and polyp~enols were used. A single substrate concentration was used for total activity determination. Almost all of the substrates were added to a final concentration of 10 m.q. For less soluble substrates, such as DL-DOPA, ( + )catechin and ( - )ep~catecbin, lower concentrations (3 mM) were used. All substrates were commercial grade (Sigma Chemical Co.) and were used without purification,

0

0.5 HA

1.0

mg I’M-’

Fig. I. Influence of increasing amounts of humic acids on tyrosinase activity. Enzyme concentration: 0.2 mg ml-‘; substrate: catechol 6 mhs. Residual activity has been calculated from relative tyrosinase activities in the presence and absence of HA. Values are the average of three determinations.

Hurnic acids-tyrosinase

1 0

50 Tyroslnasr

100

150

addrd (total units mg-’

200 HA\)

Fig. 2. Relationship between tyrosinase added and bound by humic acids. Total units of enzyme activity expressed in pm01 0, consumed min-‘. Data corrected for loss of activity from tyrosinase without HA. The results are given as a mean of three detemunations. fhe percentage of SD ranged from 2.1 to 8.7%. HA-tyrosinase

association

The isolation of the complex, formed by the association between the enzyme and HA by physical or chemical bonds, from the free components was achieved by preparing insoluble HA-tyrosinase-Ca*+ complexes (Maignan, 1982; Sarkar, 1986). By adding CaCI, to the neutral solutions containing tyrosinase in the presence of different concentrations of HA, and to the enzyme solution without HA, a coflocculation of HA and tyrosinase (“salting out” effect) and a decline in the pH to 4.5 were obtained. During the washings of the pellets with calcium acetate buffer

3500

3000

2300

2000

interactions

355

pH4.5, the tyrosinase not firmly bound to HA and the tyrosinase of the control were resolubilized (owing to the “salting in” effect) (Scopes, 1982). The insoluble washed pellets showed tyrosinase activity. The percentage of activity retained in each pellet ranged from 11.4 to 59.5% of that added, for HA: tyrosinase ratios ranging from 0.25 to 5.0 (w/w). The removal in the washings of the free enzyme and that loosely associated to the humic matrix, and the retention of tyrosinase activity in the pellets, suggest that temporary and weak binding as well as strong binding of the enzyme to the HA polymer takes place. From the difference between the total units of tyrosinase recovered in the solution and in the washings of the control and the total units recovered in the equilibrium solution and in the washings of each sample, the total units of tyrosinase bound on the HA were calculated. The results obtained are shown in Fig. 2. An HA: tyrosinase ratio ranging from 0.25 to 5.0 (w/w) was used. Binding occurred with every combination of HA and enzyme; the tyrosinase strongly bound to HA ranged from 32 to 76% of the added enzyme for HA : tyrosinase ratios increasing from 0.25 to 5.0 (w/w) respectively. The amount of enzyme stably complexed with HA was influenced by the HA concentration. Infrared analysis

Infrared spectra were recorded for tyrosinase. HA and their complexes at two HA:tyrosinase ratios (Fig. 3) and interpreted according to Bellamy (1975)

1600

Wovenumber

,600

,400

IZOO

1aJr)

q

00

0""

km-‘)

Fig. 3. Infrared spectra of tyrosinase (a), original HA (b), and their (w/w), 5: I (c) and I : I (d).

complexes.

HA: tyrosinase ratios

.

356

P.RWCXERO and V. M. -~a

and Williams and Fleming (1966). The absorption spectrum from a KBr pellet of the free tyrosinase is shown in Fig. 3(a). The vibrations due to the peptide backbone atoms involving bonded N-H stretching, primarily C%O stretching (amide I), in-plane N-H bending (amide II) and hybrid C-N stretching and in-plane N-H bending (amide III) modes, can be assigned with some confidence to the infrared lines at 3300, 1650, 1525 and 1230 cm-‘, respectively. The weak band near 3080cm-i is assigned to an additional NH absorption. The remaining lines of different intensities depend on the unknown nature of the component amino acids and may be tentatively interpreted as being attributed to C-H vibrations of -CH2 and -CH, groups (2960, 2920, 1450cm-‘), -CH, symmetrical deformation, OH deformation and/or symmetric stretching of COO- (1390cm-‘), OH defo~ation or C-O stretching of phenolic and alcoholic OH groups (1075 cm- ’ ). The principal spectroscopic features of the original HA [Fig. 3(b)] closely resemble those of the “Type III spectra” of soil HA described by Stevenson and Goh (1971). Major absorption bands occur at about 3360cm-’ (H-bonded OH), 2910 and 2840cm-’ 1710 cm-’ (C=G (aliphatic C-H stretching), stretching of carboxylic acids and ketones), 1620 cm-’ (aromatic C==C, H-bonded c--O, alkenes in conjugation with c--O or asymmetric stretching of COO-) and 1230cm-’ (C-O stretching and OH deformation of COOH). Weaker absorptions are also evident at 1390cm-’ (OH deformation and C-G stretching of phenolic OH groups, symmetric stretching of COO- or C-H deformation of aliphatic groups) and at 1040cm-’ (C-O stretching of polysaccharide-like components or Si-G vibrations of silicate impurities). The shoulder at 2600 cm-’ can be attributed to H-bonded OH in COOH, and that in the 151C-1550cm-’ region arises from aromatic skeletal c=C vibrations or N-H defo~ation (amide II). After the reaction of tyrosinase with two different amounts of HA, HA-enzyme complex compounds were produced possessing spectra shown in Fig. 3(c) and 3(d). Some indications of the types of binding occurring are appreciated by a comparative analysis of infrared spectra of the HA-tyrosinase complexes and that of the original HA. The disappearance of the absorption at 1710 cm-’ (c--O valence vibration of COOH) and of the shoulder centered at 2600cm-’ (H-bonded OH in COOH), on the one hand, and the simultaneous slight reinforcement of the band at 1390cm-i (partly due to symmetric stretching of COO-) and the significant reduction in intensity of the broad band in the 1230cm-’ region (C-O stretching and OH deformation of COOH), on the other, may be attributed to the formation of ionic bonds following proton transfer from acidic groups of HA to basic groups of tyrosinase or to electrovalent attachment of the tyrosinase to humic acid through calcium ions. Generahy, the conversion of the carboxylic groups to carboxylate ions can also be appreciated by examining the 1640-lSlOcm_’ region where stretching vibration of COO- in ionic bonds occurs. In the case of HA-tyrosinase complexes, however, a precise comparison in this spectrai region cannot be made because any modifications observed

must account for the sharp amide I and II bands of the enzyme. Changes in absorptions in the 1720-ltOOcm_’ region, more evident in the complex with the lower HA: tyrosinase ratio [Fig. 3(d)], may also be related to the partial involvement of Hbonding in the interaction between HA and the enzyme. The loss of the carbonyl absorption at 1710 cm-’ described above and the relative increase in intensity of the band centered at 1640 cm-’ may be partially attributed to the shifting to lower frequencies of the normal w stretching frequency of HA-carboxyl groups, because of H-bonding with some functional groups of the enzyme molecule. The strong vibrations of the peptide backbone groups of the complexed protein, however, make this interpretation uncertain. Possible involvement of the carboxylic and phenolic OH groups of HA to form hydrogen, other than ionic, bonds with the enzyme is suggested by the sharp reduction in intensity of the band near 1250 cm-‘. Kinetic analysis

The HA-tyrosinase complex retained its catalytic activity, thus indicating that the protein was not denatured as a result of binding. From the kinetic analysis, we obtained curves of a typical shape for enzyme systems obedient to the Michaelis-Menten equation. To compare the K,,, value of the complex with that of the free enzyme, the kinetics were carried out at pH 4.5 corresponding to the pH at which the complex was obtained. The K,,, values were calculated from the three most commonly used linear transformations of the Michaelis-Menten equation, LineweaverBurk, Eadie-Hofstee, Hanes-Woolf plots. The correlation coefficients were 0.9973, -0.9968 and 0.9915, respectively. The K,,,value obtained by the Eadie-Hofstee plot is shown in Table I. The Eadie-Hofstee plot is the most sensitive graphical technique used for detecting deviations from Michaelis-Menten kinetic (Dowd and Riggs, 1965; Walter, 1974). The mean values obtained are shown in Table I. The specific effect of HA on the K, of bound tyrosinase was substantiated by the results obtained. HA slightly increased the K,,, value of tyrosinase. The effect of HA on the I’,, of tyrosinase was not known because it was not possible to determine the protein bound to the complex and to express the V,,, values as specific activities.

Table 1. Michaelis constant and stability of the HA-tyrosinasc complex as compared lo free enzyme FVX K: (m.H) Stability toward

proteolysisb Tbennal stabilityC at 31°C at 4S’C at WC

0.56

Complexed 0.88

41

39

96 29 12

56 6 0

‘The kinetic analysis was carried out at pli 4.5. The K, value was determined by analysis of Eadic-Hofstee plot (I = -0.9968). bPercentage activity retained after incubation with protcase for 24 h. %rccntage activity retained after heating for 2 h at given temperature.

Humic

acids-tyrosinasc interactions

357

Tabk 2. Substrate specificity of free and complexed tyrorinase Relative activity’ (%) Substrate Catechol CMethylcatechol DL 3.4-Dihydroxyphenylalaninc Caffeic acid Chloropnic acid Rotocatcchuic acid (+)Catnhin (-)Epicatcchin Gallic acid

Concentration (nw) 10 10 3 IO 10 IO 3 3 IO

FrCC 100 46 19 70 21 5 23 33 0

Comckxcd 100 65 22 80 27 0 29 39 0

*Activity is relative to that of catcchol oxidation. Values arc the avenge of three determinations.

Stability of HA-tyrosinase complex The stability of the HA-tyrosinase complex toward chemical and physical denaturation (incubation with protease, exposure to elevated temperature) was compared to that of the free enzyme (Table I). After exposure to protease for 24 h, residual activity of the free enzyme was about 10% greater than that of the complexed form. The results obtained from the incubation of the HA-tyrosinase complex and of the free enzyme for 2 h at three different temperatures, point to some differences in thermal stability between the two, with the complexed form showing higher sensitivity to increasing temperatures than the free enzyme. Substrate selecthity Some diphenols and polyphenols were tested in a study of substrate specificity using both the soluble tyrosinase and the humic-enzyme complex. The data (Table 2) should be interpreted only as an indication of relative differences among the substrates, because the v,., and the degree of saturation of each substrate was not known. The physical and chemical interactions between the polyanionic support and the enzyme and between the complex and the substrate, must affect the properties of the humic-tyrosinase complex. Thus, the activity of the humic-enzyme complex toward substrates of different molecular weight and charge is likely to be different from that of the free enzyme. The relative activity, measured as a percentage of the activity toward catechol, shows that the humic-enzyme complex had a higher substrate specificity toward 4-methylcatechol and caffeic acid than the free enzyme. More slight differences of specificity can also be appreciated toward the other substrates. However, the results clearly demonstrate that no difference of substrate selectivity exists between the free and complexed enzyme. The catechol is the best substrate either for tyrosinase or for HA-tyrosinase complex, followed by caffeic acid, 4-methylcatechol, (-)epicatechin, (+ )catechin, chlorogenic acid and D,L 3,4-dihydroxyphenylalanine. Protocatechuic and gallic acids were not oxidized. DISCUSSION

The results provide information concerning the interactions between mushroom tyrosinase and HA extracted from soil. Slight inhibition of tyrosinase activity, by increasing concentrations of HA, has

been obtained using catechol as the substrate. This result is in contrast to that of Ladd and Butler (1969a). who found that HA had no effect on tyrosinase activity. However, they used experimental methods (substrate, enzymatic assay, HA:enzyme ratios) different from those we adopted in our study. Inhibition is probably due to the combination of HA with the enzyme, rather than with the substrate. Evidence that the HA combines strongly with the enzyme comes from the isolation of an insoluble well-washed HA-tyrosinase-Ca’+ complex, showing enzyme activity. The activity detected in the pellets is lower than that calculated by the difference between the total units of the supematant and washings of the control and the total units of the supematant and washings of each sample. This is not surprising, because complexed enzymes generally show reduced activity for many reasons. Some of them are the possible modification of the tertiary structure, the masking of the active site and the restriction of substrate access (Bums, 1986). Other reasons, rising from our investigation, are the inhibitory effect of HA on the tyrosinase activity and the differences of pH values of the enzymatic assays (pellets, pH 4.5; supernatants and washings, pH 7.0). The amount of enzyme, as total units, bound to the HA, depends on the HA concentration and probably on the pH of the system. The addition of CaCI, decreased the pH to a value (4.5) near to the isoelectric point of the tyrosinase. The binding of the enzyme by HA, is likely affected by pH. The isoelectric point of the tyrosinase (pH 5.0) and the pH attained after the flocculation of the interacting molecules (4.5) suggest an increase of the amount of enzyme bound on the HA. Studies on the formation of clay-protein complexes show that the adsorption of protein is normally increased as the clay suspension pH approaches the protein isoelectric point (Albert and Harter, 1973). Adsorption of protein below the isoelectric pH is also increased by the addition of an electrolyte (Armstrong and Chesters, 1964). Moreover, the decrease of pH affects the shape and particle arrangement of HA (Senesi et of., 1977), and calcium ions are believed to form complexes with the acid groups of HA and to alter the electron structure of the HA particles (Pflug and Ziechmann, 1981). Presumably, these effects influence the formation of HA-tyrosinase complex and its activity in our system. The several washings, with pH 4.5 buffer, of the pellets might cause a change in the configuration of the humic acid (Ghosh and Schnitzer, 1980) and release a fraction of entrapped enzyme.

358

P. Ruoorzao and V. M. RADOGNA

However, the removal, by washings, of the free and adsorbed enzyme from the complex, suggests that stable physical entrapment within the macromolecular matrix of the HA or chemical bonds are involved. More extended information on different binding mechanisms of such humic-enzyme association can be obtained by careful examination of the results from infrared analysis. The existence of ionic bonds which link carboxylic groups of HA to some basic groups of the enzyme or to the enzyme molecule through calcium ions, is shown by the loss and apparent reduction of the characteristic infrared absorption bands, and by the contemporaneous reinforcement of the carboxylate ion bands. The infrared analysis also demonstrates the possible involvement of carboxylic, as well as phenolic, groups of HA in weaker hydrogen bonds. The wide interference of the vibrations from covalent bonding, due to the complex chemical nature of the interacting humic and tyrosinase molecules, does not allow us to ascertain the occurrence of covalent bonds in the binding mechanisms. Knowledge of the nature of the interactions between humic colloids and enzymes is generally rather limited. The infrared results here obtained seem to indicate that, according to Sarkar and Burns (1984). adsorption and entrapment of the enzyme on or in the humic matrix appear insignificant, while the formation of ionic and hydrogen bonds in the interaction process between HA and tyrosinase is feasible. Rowell et al. (1973), however, from nitrogen content and color of soluble complexes. found that covalent and hydrogen bonds, but not ionic bonds, were important in the binding of the enzymes to a benzoquinone polymer used as an analogue of HA. The model provided here of HA-tyrosinase complex has some similar and dissimilar features when compared with data obtained from the free enzyme. The substrate selectivity is qualitatively similar to the free enzyme, but the Michaelis constant and stability to proteolysis and temperature are different. The tyrosinase complexed by the HA showed a higher K, value than that of the free enzyme and it was less thermally stable and apparently more sensitive to proteolysis than the free tyrosinase. The K,,, value is quite independent of the enzyme concentration and is widely used in enzyme kinetics as an appropriate measure of the affinity to the substrate. Although the K,,, may be considered a characteristic constant for a particular enzyme, under a certain set of conditions in a solution, its value is generally affected by the immobilization on soil components, such as HA, or on synthetic supports. Usually, more or less marked effects can be observed, but generally K,,, values of the immobilized enzymes increase (Batistic et al., 1980; Cacco and Maggioni, 1976; Nannipieri et al., 1978; 1982a; Ruggiero and Radogna, 1984; Sarkar and Bums, 1984). Causes are due to conformational changes in the tertiary structure of the enzyme and to diffusion, charge and steric effects (Ladd and Butler, 1975). The thermostability of the enzymes is generally enhanced by their complexation on soil colloids (Ladd and Butler, 1975; Rowe11 et of., 1973; Sarkar and Bums, 1984). It is unclear why the HAtyrosinase complex is less stable than the free enzyme. A similar result has been observed for glucose oxidase

adsorbed to clays (Morgan and Corke, 1976) and for urease immobilized on a clay-organic complex (Boyd and Mortland, 1985). Certainly the microenvironment around the bound enzyme must be different and it afTects the behavior of the tyrosinase toward temperature. The increase of temperature has apparently determined a change in the HA shape, thus leading to blockage of the active site of the enzyme. Moreover, a possible release from the HA at high temperature of inhibitors of the enzyme, cannot be excluded. Resistance to protease is frequently reported for humic-enzyme complexes (Pettit et al., 1976; Nannipieri et al., 1982b; Sarkar and Bums, 1984). However, the complexes may also be more sensitive to protease attack than the free enzyme (Boyd and Mortland, 1985). The possible adsorption or binding of protease on the HA, and the inhibition of the protease activity by the HA (Ladd and Butler, 1969b), are effects which may influence the stability of the humic-tyrosinase complex toward proteolysis. There is no evidence for assuming that HA influence the properties of the tyrosinase by a basically similar mechanism. Tyrosinase has copper as part of the active site structure, and the well-known ability of HA to complex metal ions (Senesi et al., 1986) might explain some of the observed effects. On the other hand, the similarity in substrate specificity of the free and complexed enzyme suggests that the mode of binding was such that there was a minimum of interference with the active site. HA may also inhibit oxidation of catechol by causing conformational changes in the enzyme, resulting in a decreased affinity of tyrosinase for its substrate. The specific properties of the HA-bound enzymes are a characteristic example of the many peculiar HA-enzyme interactions and are another illustration of the complex nature of processes that may occur in the soil microenvironment. Acknowledgernears-We thank Rosaria Mininni for skilful technical assistance. This work was partially supported by the Italian National Research Council.

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