Continuous Spectrophotometric Method for ...

Continuous Spectrophotometric Method for Determining Monophenolase and Diphenolase Activities of Pear Polyphenoloxidase ´ N VARO ´ N, JOSE´ TUDELA AND JUAN CARLOS ESPIŃ, MERCEDES MORALES, RAMO ´ NOVAS FRANCISCO GARCIÁ-CA

ABSTRACT A continuous spectrophotometric method was based on the coupling reaction between 3-methyl-2-benzothiazolinone hydrazone (MBTH) and the quinone product of the oxidation of p-hydroxyphenyl propionic acid (PHPPA) or 3,4-dihydroxyphenyl propionic acid (DHPPA) in the presence of polyphenol oxidase. The monophenolase activity of pear PPO was characterized for the first time. Solubility and stability of the adduct formed at the optimum pH (4.3) enabled the system to reach steadystate, making it possible to determine monophenolase activity with short lag periods. This, together with the value of ε for the MBTH-quinone adduct, makes this method more sensitive than other continuous methods for assaying monophenolase and diphenolase activities. Key Words: pear, polyphenoloxidase, monophenols, diphenols, MBTH

INTRODUCTION ENZYMATIC BROWNING in fruits and vegetables is often undesirable and responsible for unpleasant sensory qualities and losses in nutrient quality. Prevention of this reaction has long been studied by food scientists (Ponting, 1960; Matheis, 1987). The main enzyme involved is tyrosinase or polyphenol oxidase (EC 1.14.18.1; PPO), which has been reviewed (Va´mos-Vigya´zo´, 1981; Robb, 1984; Whitaker,1985; Mayer, 1987; Hearing and Jimeńez, 1989; Nicolas et al., 1994; Sańchez-Ferrer et al., 1995). PPO is a copper containing enzyme which, in the presence of oxygen, catalyzes two reactions: the hydroxylation of monophenols to o-diphenols (monophenolase activity), and the oxidation of o-diphenols to o-quinones (diphenolase activity). These in turn, are polymerized to brown, red or black pigments (Mason, 1955; Mathew and Parpia, 1971; Prota, 1988). Continuous spectrophotometric assays are widely used to determine the diphenolase activity of PPO. Some kinetic assays measure the appearance of quinones generated in the enzymatic reaction (Duckworth and Coleman, 1970; Garcıá-Carmona et al., 1979) or other intermediates of the melanin biosynthesis pathway, such as aminechrome (Mason, 1948) or melanochrome (Vachtenheim et al., 1985). Other methods are based on the reaction of corresponding o-quinones with coupling reagents such as ascorbate (El-Bayoumi and Frieden, 1957), NADH (Carlson and Miller, 1985), L-proline (Rzepecki and Waite, 1989), cysteine (Gauillard et al., 1993) and other chromogenic substances (Esterbauer et al., 1977; Leonowicz and Grzywnowicz, 1981; Shin et al., 1987). The solubility and stability of the reagents or products, their sensitivity and applicability to enzymes from different biological sources are problems associated with such assays. We have previously reported a continuous spectrophotometric method to determine PPO diphenolase activity using DOMA as Authors Espıń, Morales, Tudela, and Garcıá-Cańovas are with Departamento de Bioquı´mica y Biologıá Molecular-A., Facultad de Biologıá, Universidad de Murcia, E-30100 Espinardo, Murcia (Spain). Author Varoń is with Departamento de Quı´mica-Fı´sica, Escuela Universitaria Politećnica de Albacete, Universidad de Castilla-La Mancha (Spain). Address inquiries to Dr. Francisco Garcıá-Cańovas.

substrate and DOBA as chromophoric product (Rodrı´guez-Lo´pez et al., 1991). A more sensitive method used 3-methyl-2benzothiazolinone hydrazone (MBTH) to trap the enzyme generated o-quinone in PPO oxidation of L-dopa (Winder and Harris, 1991). The stoichiometry and mechanism of this reaction have been well established (Rodrı´guez-Lo´pez et al., 1994). Diphenolase activity of pear PPO has been assayed on several substrates such as catechol, 4-methylcatechol or chlorogenic acid, by measuring absorbance increase at 420 nm (Tate et al., 1964; Rivas and Whitaker, 1973; Halim and Montgomery, 1978; Vamos-Vigya´zo´, 1981; Zhou and Feng, 1991; Siddiq et al., 1994), or using catechol or chlorogenic acid as substrates and measuring oxygen consumption (Smith and Montgomery, 1985; Wissemann and Montgomery, 1985). Radiometric discontinuous methods are commonly used to assay monophenolase activity of PPO (Pomerantz, 1964; Husain et al., 1982; Winder and Harris, 1991). Such methods are very sensitive but are discontinuous, cumbersome and may require up to 30 min for each assay (Pomerantz, 1964; Husain et al., 1982; Winder and Harris, 1991). The most widely used continuous method measures formation of dopachrome at 475 nm using tyrosine as substrate (Mason, 1948), but has poor sensitivity due to the low value of ε. In the course of research into polyphenoloxidase from plant sources, we needed a measure of monophenolase activity of pear PPO which had not been previously reported. Our objective was to develop a spectrophotometric method for its characterization. This method was based on the coupling reaction between MBTH and the quinone products of the oxidation of p-hydroxyphenyl propionic acid (PHPPA) in the presence of PPO. A more sensitive method than those previously described was also developed for assaying diphenolase activity (Fig. 1). MATERIALS AND METHODS Reagents L-Dopa, dopamine hydrochloride, 4-methylcatechol, MBTH, PHPPA, DHPPA, L-dopa methyl ester and L-a-methyldopa were purchased from Sigma (USA) and catechol from Merck (Germany). All other chemicals were analytical grade and supplied by Fluka (Spain). Stock solutions of phenolic substrates were prepared in 0.15 mM acetic acid to prevent autooxidation. Both 50 mM sodium acetate (pH 3.0–5.5) and 50 mM sodium phosphate (pH 5.8–7.0) buffers were used. The acidic character of MBTH required 50 mM buffer in the assay medium. To dissolve the MBTH-quinone adducts, 2% (v/v) N,N'-dimethyl formamide (DMF) was added to the assay medium (Winder and Harris, 1991; Rodrı´guez-Lo´pez et al., 1994). Triton X-114 (TX-114) was obtained from Fluka (Madrid, Spain) and condensed three times as described by Bordier (Bordier, 1981), using 0.1M sodium phosphate buffer (pH 7.3) containing 20 mM EDTA. The detergent phase of the third condensation contained a concentration of 22% TX-114 (w/v).

Enzyme source Pears of the blanquilla cultivar picked in Murcia, Spain, at commercial maturity and stored at 57C were used as enzyme source. Pear PPO was

Volume 61, No. 6, 1996—JOURNAL OF FOOD SCIENCE—1177

MONOPHENOLASE AND DIPHENOLASE ACTIVITIES OF PEAR POLYPHENOLOXIDASE . . .

Fig. 1—Mechanism proposed to explain the oxidation of PHPPA and DHPPA by pear PPO: M: PHPPA; D: DHPPA; Q: DHPPA-o-quinone; N: MBTH; ND: MBTH-DHPPA adduct; NQ: MBTH-DHPPA-o-quinone adduct.

extracted and partially purified by a modified method using TX-114 (Bordier, 1981; Sańchez-Ferrer et al., 1989; 1993). Pears were washed and hand-peeled. A 150g sample was homogenized with 75 mL of cold buffered 0.1M sodium phosphate pH 7.3, 20 mM EDTA and 6% (w/v) TX-114 for 2 min. The homogenate was kept at 47C for 60 min before being centrifuged at 100,000 3 g for 45 min at 47C. The supernatant was collected and used as crude enzyme extract. It was subjected to temperature-induced phase partitioning by increasing the TX-114 concentration by an additional 8% (w/v) at 47C and then warming to 357C for 15 min. The solution became turbid due to the formation, aggregation and precipitation of large micelles of detergent containing hydrophobic proteins and phenolic compounds. This solution was centrifuged at 8000 3 g for 15 min at 257C. The detergent rich phase was discarded and the supernatant was subjected to an additional phase-partitioning step with of 8% (w/v) TX-114. The procedure was repeated once more to remove remaining phenols. The clear supernatant containing soluble pear PPO was acidified to pH 5.0, kept for 1 hr at 47C, and centrifuged at 100,000 3 g for 30 min at 47C. The pellet was discarded and the supernatant brought to 30% saturation with solid (NH4)2SO4 under continuous stirring at 47C. After 15 min the solution was centrifuged at 80000 3 g for 30 min at 47C and the pellet discarded. The clear supernatant was saturated to 80% with (NH4)2SO4 and stirred for 30 min at 47C. The solution was centrifuged at 100,000 3 g for 30 min and the precipitate dissolved in a minimal volume of deionized water. Salt was removed by a desalting column of Sephadex G-25. The enzyme was stored at 2307C with a 10% loss of original activity after 8 months, and no discoloration. This protocol gave a 10-fold purification of the enzyme extract which preserved its monophenolase activity. Other methods Protein content was determined by the method of Bradford (1976) using bovine serum albumin as standard. Spectrophotometric assays Absorption spectra were recorded in an ultraviolet-visible Perkin-Elmer Lambda-2 spectrophotometer, interfaced with an Amstrad PC2086 microcomputer, with 60 nm/s scanning speed. Temperature was controlled at 257C using a Haake D1G circulating bath with a heater/cooler and checked using a Cole-Parmer digital thermometer with a precision of 50.17C. Kinetic assays were also carried out with the instruments by measuring the appearance of products in the reaction medium. Reference cuvettes contained all components except the substrate, with a final volume of 1 mL. Kinetic data analysis The direct determination of parameters were reported as value 5 standard deviation, supplied by the statistic program used Sigma Plot 5.0 (Jandel Scientific, 1992). For parameters calculated from others, corresponding standard deviations were calculated considering appropiate expressions of error propagation (Endrenyi, 1981). The values of Km and Vmax on different substrates were calculated from triplicate measurements of the steady state rate, Vss, for each initial con-

centration of substrate, [S]0. Reciprocals of variances of Vss were used as weighting factors in the nonlinear regression fitting of Vss vs [S]0 data to the Michaelis equation (Wilkinson, 1961; Endrenyi, 1981). Fitting was carried out by using a Marquardt’s algorithm (Marquardt, 1963) implemented in the Sigma Plot 5.0 program (Jandel Scientific, 1992). Initial estimations of Km and Vmax were obtained from Hanes-Woolf equation, a linear transformation of the Michaelis equation (Wilkinson, 1961). Sensitivity of the method was tested with triplicate determinations of Vss for each initial concentration of PPO, [PPO]0. Reciprocals of the variances of Vss were used as weighting factors in the linear regression fitting of Vss vs [PPO]0 (Wilkinson, 1961; Endrenyi, 1981). Precision was checked by 10 assays of Vss at each of three [PPO]0 values, whose coefficients of variation were calculated. Sensitivity of the method was characterized from 10 measurements of the blank, followed by evaluation of the corresponding detection and quantitation limits (ACS, 1980).

RESULTS Diphenolase activity of PPO Formation and properties of MBTH-quinones adducts. To determine characteristics of these adducts, oxidation of various diphenols by O2 catalyzed by PPO was carried out in the presence of MBTH. The pigment formed with L-dopa, dopamine, L-amethyldopa, L-dopa methyl ester, catechol, 4-methylcatechol or DHPPA was dark pink with absorbance maximum ranging from 494 to 510 nm (Fig. 2, Table I). The pH affected solubility and stability of the adducts. Solubilization of these compounds was achieved by adding 2% (v/v) DMF to the reaction medium (Winder and Harris, 1991; Rodrı´guez-Lo´pez et al., 1994). Under these conditions, adducts formed by MBTH addition to the o-quinone from different o-diphenols were soluble at every pH (from 3.5 to 7). At pH 5 or lower, adducts were soluble and stable, whereas at higher pH adducts were unstable but showed an isosbestic point. Exceptions were the o-diphenols catechol and 4-methylcatechol. The MBTH-benzoquinone adduct showed an isosbestic point at pH 6.8 (results not shown), but at acid pH both adducts were slightly insoluble. Thus it was only possible to determine the diphenolase activity in rapid kinetic assays. However, it was not possible to determine monophenolase activity since this required long kinetic assays. MBTH is a potent nucleophile through its amino group, which differs in degree of protonation-deprotonation depending on pH. The ionization constant for this group has been determined by both potentiometric and spectrophotometric methods (pKa 5 5.8 5 0.4). The saturating MBTH concentration, [MBTH]sat, using different o-diphenols was determined by measuring the initial rate of change in absorbance at the lmax of the corresponding adduct, using different amounts of MBTH (Fig. 3). Depending on its stability, each o-quinone needs a different [MBTH]sat to complete adduct formation (Table I).

1178—JOURNAL OF FOOD SCIENCE—Volume 61, No. 6, 1996

yphenyl propionic acid (Fig. 4). Note that the VDmax and KDm followed the relation catechol . 4-methylcatechol . DHPPA, which explains why the catalytic efficiencies (VDmax/KDm (Table I) had similar values. Effect of [PPO]. Linearity was obtained between enzymatic activity and PPO concentration (Fig. 5), which might be useful as a linear calibration curve for determination of microquantities of PPO in problem samples. The method was applied in triplicate assays (Marquardt, 1963; Endrenyi, 1981) and a linear regression fitting was obtained (Fig. 4). The precision of the method was checked by repeating the estimation of VDss ten times for three samples at three levels of [PPO]0 0.11, 0.55 and 1.05 µg/mL. The corresponding coefficients of variation were 6.8, 1.6, and 1.2%. Sensitivity of the method was characterized by determining the limit of detection (LODD 5 13.5 ng/mL) and the limit of quantitation (LOQD 5 14.4 ng/mL).

l

Monophenolase activity of PPO

Fig. 2—Spectra for oxidation of o-diphenols by pear PPO in the presence of MBTH. Conditions were: 126 µg/mL PPO, 10 µM odiphenol, 2% DMF and 50 mM AB pH 4.3. Concentrations of MBTH for each o-diphenol were: (a) Catechol, 0.1 mM MBTH; (b) L-Dopa, 3 mM MBTH; (c) DHPPA, 1 mM MBTH; (d) Dopamine, 1 mM MBTH; (e) L-Dopa methyl ester, 3 mM MBTH.

The diphenolase activity of PPO was assayed on several substrates at pH 4.3, the optimum for pear PPO (cv. blanquilla). The comparison of o-diphenols, in the presence of MBTH showed lower sensitivity for L-dopa and derivatives than for catechol and derivatives (Table I). Molar absorptivities for corresponding MBTH-quinone adducts had similar values (Table I). Thus, the different values of VDmax indicated the main contribution of the respective values of the catalytic constants of PPO for each o-diphenol (Table I). When other detectable species were measured, lesser molar absorptivities were obtained: oquinones, ε ' 2000 M21 cm21 (Waite, 1976); aminechromes, ε ' 3500 M21 cm21 (Mason, 1948); ascorbic acid disappearance, ε ' 11000 M21 cm21 (El-Bayouimi and Frieden, 1957); cysteine adducts, ε ' 2500 M21 cm21 (Gauillard et al., 1993); proline adducts, ε ' 5030 M21 cm21 (Rzepecki and Waite, 1989). MBTH-quinone adducts had much higher molar absorptivities which ranged from 32500 to 52000 M21 cm21. The MBTHquinone adduct of the pair PHPPA/DHPPA had a mean value of 40000 M21 cm21 (Table I). The higher values of VDmax corresponded to catechol . 4-methylcatechol . DHPPA. The low solubility at pH 4.3 of MBTH-quinone adducts of 4-methylcatechol and catechol limited the reliability of measurements, especially when their monophenols were studied. Since the MBTH-quinone adduct of DHPPA showed high sensitivity, good solubility and stability, DHPPA was chosen as the substrate for PPO diphenolase activity. Effect of [D]0. The effect of the initial concentration of different o-diphenols, [D]0, on their rate of oxidation catalyzed by PPO, VDss, in the presence of MBTH, was kinetically characterized. A hyperbolic dependence of VDss on [D]0 was obtained for each o-diphenol considered (data not shown). These data were fitted by non-linear regression to the Michaelis equation, giving the KDm values (Table I). Dopa and its derivatives showed lower KDm values than catechol and its derivatives. The respective VDmax values were described in µM/s (Table I). The ratio of VDmax was of note because it was much higher for catechol and its derivatives, especially 3,4-dihydroxyphenyl propionic acid (Table I). A mean value of VDmax 5 3.96 µM/s was obtained with 12.5 µg/mL PPO when assayed on 3,4-dihydrox-

The MBTH method was broadly applicable to the different monophenols tested. Scarce activity was detected with L-tyrosine, L-a-methyltyrosine or L-tyrosine methyl ester. Slight activity was detected with phenol and p-cresol after long assay times but their MBTH-quinone adducts were unstable. Since DHPPA had been selected as the best o-diphenolic substrate for pear PPO, its corresponding monophenol was used in further monophenolase assays. The high speed of the nucleophilic addition of MBTH resulted in quick regeneration of o-diphenol (Fig. 1) and a shortening of the lag period (Fig. 6). Another reason for using PHPPA in assaying the monophenolase activity of PPO was its high ε (Table I) and the good solubility and stability of its MBTH-quinone adduct. Effect of [M]0. Kinetic characterization of the monophenolase M activity of PPO on PHPPA gave KM m 5 0.5 mM and Vmax 5 0.07 µM/s with 71.5 µg/mL PPO (Fig. 4), while tyramine M yielded a KM m value of 1.7 mM and Vmax of 5 nM/s. Thus, the monophenolase activity of PPO was 14 times higher on PHPPA than on tyramine. The lower apparent catalytic constant of monophenolase activity of PPO led to higher values of LODM and LOQM than in the case of diphenolase activity, LODD and LOQD. Effect of [PPO]0. As with diphenolase activity, monophenolase activity was linear with PPO concentration (Fig. 5). The precision of the method was checked by repeating the estimation of VM ss ten times for three samples at three levels of [PPO]0 0.49, 2.45 and 4.42 µg/mL, the corresponding coefficients of variation being 5.3, 2.3, and 1.4%. The sensitivity of the method was characterized by determining the limit of detection (LODM 5 32 ng/mL) and the limit of quantitation (LOQM 5 41 ng/mL). DISCUSSION Diphenolase activity The assay involving oxidation of PHPPA and DHPPA by PPO in the presence of MBTH has been improved. Diphenolase activity of pear PPO has long been determined using catechol, 4-methylcatechol or chlorogenic acid as substrates (Tate et al., 1964; Rivas and Whitaker, 1973; Halim and Montgomery, 1978; Vamos-Vigya´zo´, 1981; Zhou and Feng, 1991; Siddiq et al., 1994). Activity was measured following the change in absorbance at 420 nm or oxygen consumption (Smith and Montgomery, 1985; Wissemann and Montgomery, 1985). These methods measure total activity of the enzyme, although they have some disadvantages such as the low ε for o-quinones and the high instability due to the rapid evolution through intermolecular reactions. The method we propose, based on the coupling reaction between MBTH and the o-quinones, measures half the activity (Fig. 1) but is much more sensitive because of the high ε for the MBTH-quinone adducts. The substrates which yielded the highest VDmax (µM/s) values were catechol, 4-methylcatechol and DHPPA. The first and sec-


MONOPHENOLASE AND DIPHENOLASE ACTIVITIES OF PEAR POLYPHENOLOXIDASE . . .

Fig. 3—Determination of saturating MBTH concentration in the oxidation of o-diphenols by pear PPO. Conditions were: 2 % DMF, 50 mM AB pH 4.3 and: ( n ) 2.1 mM L-Dopa, 9 µg/mL PPO; ( l ) 20 mM Catechol, 0.6 µg/mL PPO; ( ● ) 5 mM DHPPA, 0.5 µg/mL PPO.

Fig. 4—Effect of PHPPA and DHPPA concentration on monophenolase and diphenolase activities of pear PPO, respectively. Conditions were: 2% DMF, 50 mM AB pH 4.3, 1 mM MBTH and ( C ) Monophenolase activity: (0.1–5 mM) PHPPA; 71.5 µg/mL PPO. ( ● ) Diphenolase activity: (0.5–15 mM) DHPPA; 12.5 µg/mL PPO.

ond gave adducts with solubility problems, especially when their monophenols were used in long kinetic assays. Therefore, DHPPA was chosen as the optimum substrate for pear PPO.

method enables determination of both PPO activities for the first time. This was not possible with any of the other substrates tested. NOMENCLATURE

Monophenolase activity The monophenolase activity of pear PPO has never been published, perhaps because of the lack of sensitive and reliable assays. When substrates with an aminoacidic chain, (L-tyrosine, L-a-methyltyrosine or L-tyrosine methyl ester) were used, little activity was detected. Slightly higher activity was detected with substrates with a shorter chain such as phenol and p-cresol, after long times. However, these measurements were not reliable due to low solubility of the corresponding MBTH-quinone adducts, which became more notable at prolonged assay times. A sensitive and reliable measurement of PPO monophenolase activity was obtained with PHPPA. When results from the different substrates were compared, the pair PHPPA/DHPPA was optimum for measuring activity of pear PPO.

D [D]0 DHPPA DMF DOBA DOMA EDTA KDm KM m LODD LODM LOQD LOQM

CONCLUSIONS OUR METHOD FOR DETERMINATION of monophenolase and diphenolase activity of pear PPO has several advantages over other continuous spectrophotometric methods. The substrates, PHPPA and DHPPA, showed no solubility problems. The MBTH-quinone adduct was stable at the optimum pH for pear PPO and showed a high molar absorptivity. The method was ten times more sensitive than when other substrates were used, mainly due to the high value of the catalytic constant of PPO toward DHPPA. The

M [M]0 MBTH [MBTH]sat PHPPA PPO [PPO]0 [S]0 t

o-Diphenol (DHPPA) Initial o-diphenol concentration 3,4-Dihydroxyphenyl propionic acid N,N'-Dimethylformamide 3,4-Dihydroxybenzaldehide 3,4-Dihydroxymandelic acid Ethylenediaminetetraacetic acid Km value for o-diphenol (DHPPA) Km value for monophenol (PHPPA) Limit of detection of the diphenolase activity of pear PPO Limit of detection of the monophenolase activity of pear PPO Limit of quantitation of the diphenolase activity of pear PPO Limit of quantitation of the monophenolase activity of pear PPO Monophenol (PHPPA) Initial monophenol (PHPPA) concentration 3-Methyl-2-benzothiazolinone hydrazone Saturating MBTH concentration p-Hydroxyphenyl propionic acid (Pear) polyphenoloxidase Initial PPO concentration Initial substrate concentration Lag period

Table 1—Properties of MBTH adducts from several o-diphenols and comparison of diphenolase activitya

o-Diphenol L-Dopa Dopamine L-a-Methyldopa L-Dopa methyl ester Catechol 4-Methylcatechol DHPPA

lmax

ε (M21 cm21)

[MBTH]sat (mM)

VD max (µM/s)

D (mM) Km

21 4 D VD max/Km (s ) 3 10

Ratio of ε

Ratio VD max

507 503 504 504 500 494 500

38000 42500 52000 52000 32500 32500 40000

3 1 1.5 3 0.1 0.2 1

0.11 0.27 0.04 0.05 13.71 6.55 3.96

3.15 1.45 1.93 0.93 16.44 8.13 4.93

0.35 1.86 0.21 0.53 8.34 8.05 8.03

1.17 1.30 1.60 1.60 1 1 1.23

2.01 5.40 1 1.30 205.40 98.10 73.11

a The assay medium was 50 mM acetate buffer, pH 4.3, with 2% DMF. The values of ε for the MBTH adducts were determined by fast oxidation of low concentrations of

diphenols in the presence of high concentrations of PPO with saturating MBTH ([MBTH]sat).


o-

Fig. 5—Calibration curve for the determination of pear PPO. Conditions were: 2% DMF, 1 mM MBTH, 50 mM AB pH 4.3. ( ● ) Diphenolase activity, 50 mM DHPPA. ( C ) Monophenolase activity, 4 mM PHPPA.

TX-114 VDm VM m Vss VDss VM ss

Triton X-114 Maximum rate of the diphenolase activity of pear PPO Maximum rate of the monophenolase activity of pear PPO Steady state rate Steady state rate of the diphenolase activity of pear PPO Steady state rate of the monophenolase activity of pear PPO REFERENCES

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Zhou, H. and Feng, X. 1991. Polyphenol oxidase from Yali pear (Pyrus bretschneideri). J. Sci. Food Agric. 57: 307–313. Ms received 12/9/95; revised 2/27/96; accepted 4/10/96.

This paper was partially supported by a grant from the Direccioń General de Investigacioń Cientı´fica y Tećnica (Spain), project number DGICYT PB92-988, as well as by the Consejerıá de Cultura y Educacioń de la Comunidad Autońoma de Murcia (Spain), project number PCT94/95. Juan Carlos Espıń had a fellowship from the Plan Nacional de Formacioń de Personal Investigador (Spain), reference AP93 34785457.
