J Sci Food Agric 1993,62, 245-252

Effect of Alkali Halides on a-Chymotrypsin Activity in the Plastein Reaction Pedro Lozano* and Didier Cornbest Dbpartement de Genie Biochimique et Alimentaire, UA-CNRS No. 544, Institut National des Sciences Appliquies, Complexe Scientifique de Rangueil, 3 1077 Toulouse Cedex, France (Received 2 November 1992; revised version received 5 March 1993; accepted 23 April 1993)

Abstract: The influence of different alkali halides (LiF, LiCI, LiBr, NaF, NaC1, NaBr, KF, KCl and KBr) on a-chymotrypsin-catalysed plastein synthesis has been studied in aqueous medium at different substrate concentrations. The results showed an enhancing effect on the plastein synthesis enzyme action by the presence of salts, proportional to salt concentration, which was decreased when the substrate concentration was increased. Additionally, these facts allowed the ions to be classified as a function of their activation power (F- > C1- > Br-; K+ > Na+ > Li+), which was in agreement with the interpretation of the Hofmeister lyotropic series. The influence of the several phenomena produced by the presence of salts in the plastein reaction, such as, salt-substrate interactions and water activity, as well as the presence of deactivated enzyme was also analysed. The obtained results showed that the substrate concentration is the most important factor, and the activating effect of salts could be simultaneously involved with both a structural change on the substrate conformation and a reduction of water activity into the reaction media, enhancing the catalytic capability of the a-chymotrypsin towards a peptide synthesis action.

Key words : a-chymotrypsin, salts, water activity, plastein reaction, peptide synthesis, peptide condensation, transpeptidation.

INTRODUCTION formation by a reversal of the usual peptide bond hydrolysis catalysed by a protease, to produce ' resynthe-

Salts affect in many different manners the properties of sised' protein or polypeptides from an hydrolysate of macromolecules such as their stability, solubility and protein (Arai et al 1981), should be included in this catalytic capability (Combes et al 1988). At low salt context. This process is of considerable interest for the concentration, stabilising effects are due to specific food industry, such as for the debittering of hydrolysed interactions between the salt cations and proteins. At protein from unconventional sources, offering a potential high concentration, the salts produce effects depending means of supplementing food proteins with the desired on both the concentration and the nature of the salt, amino acids and controlling the functional properties of resulting in either the stabilisation or the denaturation of the polypeptides in food materials (Fujimaki et a1 1977). proteins, as well as their salting-in or salting-out (Schmid The plastein reaction requires that three conditions are 1979). Additionally to the effect on protein stability, the present: (i) a low molecular weight for the peptidic presence of salts in an hydrolytic enzyme-catalysed substrate, (ii) a substrate concentration in the reaction reaction mixture, decreases the water activity and, medium between 20 and 40 YO (w/v), and (iii) an optimum consequently, can shift the equilibrium towards the pH value for the 'synthetic' activity of the protease. The synthetic reaction (Hahn-Hagerdal 1986). The plastein role of proteolytic enzymes and their kinetic mechanism reaction, which is considered to involve polypeptide in the plastein reaction has been very controversial.

Several authors (Yamashita et al 1970; Tsai et al 1974;

Molecular B e Inmunologia, Facultad de Quimica, Universidad de Murcia, Campus de Espinardo, E-30001 Murcia, Spain. dation mechanism for the plastein reaction, while other $ To whom correspondence should be addressed. authors (Determann et a1 1965; Yamashita et a1 1973)

J Sci Food Agric 0022-5142/93/$06.00 0 1993 SCI. Printed in Great Britain

* Present address: Departamento de Bioquimica Y Biologia Hofsten and La]asidis 1976) have postulated a transpepti-

245

246 P Lozano, D Combes

have considered it as a reversal of normal hydrolytic reactions, occurring by a condensation mechanism. In a previous paper (Lozano & Combes 1991), the authors concluded that the a-chymotrypsin action on the plastein synthesis presents a high dependence on the micro- environmental conditions (substrate concentration). Depending on these conditions, a general mechanism was postulated where both transpeptidation and con- densation enzymic pathways are present.

The purpose of this work was to ascertain the role of additives, such as salts, on the a-chymotrypsin action on the plastein synthesis. Tanimoto et a1 (1975) studied the effect of several salts on plastein formation by a- chymotrypsin from a subtilisin BPN’ hydrolysate of ovalbumin. This study showed that the presence of salts enhanced plastein yield to a maximum level of lo%, when salt concentration was 1 M. In the case of NaCl, these authors obtained two maximal levels of activity for the assayed salt concentrations (0.1 and 0.8 M, respect- ively), and concluded that the first maximum resulted from an increase in enzyme activity, and the second maximum was ascribed to the salting-out of the product due to the high concentration in NaCl.

The aim in this paper is to establish a relationship between the environmental conditions of the reaction, defined by the type of salts, salt concentration, water activity (Aw) and substrate concentration, and the synthesis of plasteins for their possible exploitation in the use of proteins in novel food applications.

In this four-component system : enzyme-substrate- water-salt, the knowledge of the preferential interactions between the proteins and the solvent components will explain the manner in which additives affect solubility, stability and activity of proteins. The effect of salts on solubility and stability of proteins has been extensively described in the literature ; different methodologies have been used, such as the measurements of the salt molal surface increment (Melander and Horvath 1977) or the balance between protein hydration and salt binding (Arakawa and Timasheff 1982, 1984). The mechanism of protein salting-in and salting-out, as well as, the dynamics of water (Terpstra et aZ1990), or the protective effect on the enzymic activity (Combes et a1 1988; Ye et a1 1988) produced by a high salt concentration media have been analysed. In the present case, a pepsin hydrolysate of albumin (mol wt < 10 kDa) was used as standard substrate. As previously reported (Lozano and Combes 199 1) the plastein reaction is enzyme-catalysed and the viscosity of the reaction mixture increases with the plastein synthesis. This increase is proportional to the enzyme concentration which also affects the secondary bonds interactions (between substrate, product and enzyme) involved in the plastein precipitation. Then, the enzyme concentration was chosen in order to minimise the effect on the rheological phenomena. The high viscosity due to substrate concentration and the presence of a salt, implies that the physico-chemical properties of

water will play an exceedingly important role, changing the substrate conformation, as well as, affecting the function of a-chymotrypsin in the plastein reaction media.

MATERIALS AND METHODS

Bovine serum albumin (Sigma) was used for the preparation of substrate.

Pepsin (EC 3.4.23.1) from porcine stomach mucosa was used as the hydrolysing catalyst to obtain the substrate, and a-chymotrypsin (EC 3.4.21 . 1) from porcine pancreas was used to catalyse the plastein reaction. Both enzymes were obtained from Sigma.

All remaining reagents were analytical grade

Substrate preparation

A 10% (w/v) albumin water solution (pH 1.6) was treated by pepsin (1 YO w/w) with magnetic stirring for 48 h at 40°C. The reaction was stopped by increasing the pH to 7.0 with NaOH, and the resulting hydrolysate solution was ultrafiltered through polysulphone mem- branes (lO000Da cutoff) and then concentrated in a Rotavapor to 55% (w/v). The degree of hydrolysis of this substrate was 84% and the free amino groups concentration increased from 0.66 to 1.75 pmol NH3+ per mg of albumin (Lozano and Combes 1991).

Plastein reaction

Into an Eppendorf tube of 1 ml total volume, amounts of 100, 200 or 350 mg of the concentrated hydrolysate of albumin were placed, and 3.5 mg of a-chymotrypsin were added. The reaction volume was adjusted to 1 ml by addition of water or aqueous salt solution, and the reaction mixture incubated without stirring at 40°C and pH 7 (Lozano and Combes 1991). Aliquots of 50 pl were extracted from the reaction mixture, previously homo- genised by shaking, at different times for plastein product quantification.

Determination of plastein activity

Aliquots of 50 p1 were mixed with 950 pl of a 10.53 YO (w/v) trichloroacetic acid solution (TCA), and then introduced in an ice bath to quench the reaction. TCA- treated samples were centrifuged (1 0 min at 2800 x g ) to separate the protein precipitate. This precipitate was redissolved in a 50% (v/v) acetic acid solution and spectrophotometrically quantified at 280 nm. Plastein products and plastein yield were determined as follows :

plastein products : P, = P, - Po where P, is mg of plastein products at time t, P, is mg of protein precipitate at time t , and Po is mg of protein precipitate at time 0.

plastein yield (%) = P,/S,, x 100 where So is mg of substrate at time 0.

Alkali halides in plastein reaction 241

One unit of plastein activity was defined as the amount of enzyme that produced 1 mg of plastein per min under optimum assay conditions (Lozano and Combes 1991).

Gel permeation chromatography

Gel permeation of the plastein reaction mixture was carried out in a fast protein liquid chromatography (Pharmacia Fine Chemical) using a column (10 x 300 mm) of Superose @12 (Pharmacia Fine Chemi- cal), equilibrated and eluted with 50 YO (v/v) acetic acid at a flow rate of 0.1 ml min-l. An aliquot of the reaction mixture (15 pl) was dissolved in 185 pl of the eluent and applied to the column. The absorbance of the column effluent was monitored at 280 nm (Lozano and Combes 1991).

Determination of A ,

A , was determined using a humidity and temperature digital indicator, HUMIDAT-IC I1 (Novasina), with a humidity sensor model BS-3(4)/PP (Novasina) at a temperature of 20°C. The humidity sensor was checked and periodically recalibrated at three points, with control saturated salt solutions (LiCl, A , = 0.1 13; Mg(NO,),, A , = 0544; and BaCl,, A , = 0,905) for the overall measuring range.

Amino acid analysis

A lyophilised sample (1 mg) was hydrolysed in a Pico- Tag system (Waters) as follows: first, the system removed exhaustively the air by a repeated freeze-thaw technique and then, the hydrolysis was carried out in a 6 M HC1 atmosphere at 110°C for 24 h. Later, the liberated amino acids were determined with an AminoQuant (Model 1090 Series 11, Hewlett-Packard) automatic amino acid analyser, using both o-phtalaldehyde and 9- fluorenylmethyl chloroformate pre-column derivatisa- tion methods for the spectrophotometric quantification.

RESULTS AND DISCUSSION

The influence of different alkali halides (LiF, LiC1, LiBr, NaF, NaC1, NaBr, KF, KCl and KBr) on the plastein synthesis catalysed by a-chymotrypsin was studied at different concentrations. As previously reported (Lozano and Combes 1991), the a-chymotrypsin action exhibits a high dependence on substrate concentration in the plastein synthesis. Thus, three different values of this parameter (10, 20 and 35 YO w/v) were chosen as

representative conditions of different microenvironments in the enzyme action, for the study of the influence of salts. The effect of these alkali halides has been analysed separately by comparison of the different salt-activity profiles, as a function of the halide or the alkali ion, the substrate conformation and the dynamic of water for the different substrate concentrations (Waks 1986). The assayed concentrations of salts were determined by their solubility in the reaction media.

Figure 1 shows the evolution of a-chymotrypsin activity in the plastein synthesis, at three different substrate concentrations, as a function of the alkali halides concentration. As can be seen, the increase in the salt concentration generally induced an enhancement of the catalytic capability of the a-chymotrypsin. This activation effect on the plastein synthesis was attenuated when the substrate concentration was simultaneously increased. However, LiBr was the less effective activating agent being a deactivating agent at the highest con- centration assayed, while both the sodium and potassium salts were activators, increasing the synthetic activity between 1.2- and 3.2-fold. Thus, in agreement with the results, it could be directly concluded that the presence of salts in the plastein reaction media generally increases the catalytic capability of the a-chymotrypsin. This increase is roughly proportional to their concentration except for Lit. Furthermore, for a better understanding of the mechanism of action of these additives, these results should be analysed separately, as a function of the particular effect of each salt on each component of the medium : enzyme-substrate-solvent.

First, in order to know the particular effect of the salt on the enzyme action, the slopes of these activity-salts concentration profiles were determined and depicted as a function of the assayed ion. For all substrate concen- trations, this slope-ion graphic was similar, and Fig 2 reports the results obtained when a 20 % (w/v) substrate concentration was used. Then, if the sodium halides behaviour, is compared, it is found that the activation effect decreased (F- > C1- > Br-) when increasing the anion size (F- < C1- < Br-). On the other hand, in the series of alkali chlorides, the opposite effect was observed: the increase of the cation size (Lit < Na+ < K+) enhanced the catalytic power of the a-chymotrypsin in the plastein reaction (Li+ < Na+ < K+). Moreover, this effect of salts was shown to be additive: when the higher anion (Br-) and the smaller cation (Li+) are assayed together (LiBr), a negative effect on the a- chymotrypsin action was observed, while in their chloride (LiCl) and potassium (KBr) salts, respectively, they were neutralised by the activating power of these ions (Cl- and K+). Ions may affect both the activity of the enzyme and the solubility of the reacting species (substrate, enzyme, product). As no specific effect of alkali halides on a- chymotrypsin activity has been described, the positive effect of the assayed ions on the plastein reaction may be attributed to their influence on the hydrolysis/synthesis

248 P Lozano, D Combes

A

bQ v 50

[S] = 20 % w/v [S] - 20 % w/v

5 250 [S] - 35 I w/v 011 [s] = 35 x w/v

I [S] - 10 I w/v

[S] - 20 I w/v I

Fig 1. Effect of alkali halides concentration on the plastein reaction catalysed by a-chymotrypsin, using a peptic hydrolysate of albumin as standard substrate, at three different concentrations (10, 20 and 35% w/v) in optimum conditions of assay (40°C;

pH 7.0).

m Na+

U

F- a- B T

Fig 2. Overall dependence of the catalytic activity of a- chymotrypsin in the plastein synthesis on the ions present in the

reaction media.

equilibrium of the plastein production pathway (Hahn- Hagerdal 1986). Several authors (Melander and Horvath 1977; Schmid 1979; Combes et aZl988; Przybycien and Bailey 1989; Franks, 1991) have carefully described the effect of salts on protein stability and solubility: these effects could be related to the Hofmeister lyotropic series. Anions and cations of these series are classified in agreement to their salting-in (Br-, K+) or salting-out (F-, Li+) effect. In this case, where both substrate and enzyme are susceptible to be affected by the presence of the salts,

activation by non-specific salt-a-chymotrypsin inter- actions or conformational changes on the polypeptide chain of substrate ([S] was 3&100 times higher than [El), should be responsible for the enhancement on the a- chymotrypsin action in the plastein synthesis.

Melander and Horvath (1977) have developed the current salting-out theory, where the effect of neutral salts on electrostatic and hydrophobic interactions in precipitation and chromatography of proteins is dis- cussed. In their paper, the corresponding hydrophobic energy change produced by the presence of salts is evaluated from the non-polar contact area of the interacting species and surface tension of the medium, quantified by its molal surface tension increment. This parameter forms the basis of a natural lyotropic series, studied by using 44 different salts, and in the case of the alkali halides, is more increased by the reduction of the anion size than by the increase in the cation size. As can be seen, our results are in agreement with this theory. Additionally, Przybycien and Bailey (1 989) have recently reported a thorough study of the effect of the lyotropic series salts (Na,SO,, NaCl, NaBr, KBr and KSCN), as precipitating environments, on recovering activity of a- chymotrypsin. These authors showed that the fraction of precipitate, which is active upon dissolution, is a function of the salt molal surface tension increment, and the specific activity of precipitate remained essentially con- stant regardless of salt concentration type. They con- cluded that the protein denaturation in precipitation is an ' all-or-nothing' phenomena, where the enzymic

Alkali halides in plustein reaction

A

249

0 Control [El : [S] = 1 % w/l 0 [Ed] = 1 % w/w

~ A [Ed] = 2 % w / w [S] = 35 % w/v

30

25

20

n

X n 10 0

3 '5

.- w 5

.- 3 0

2 > .-

25

20

15

10

5

+

(O,.,V) 10% w/v (r,0.*) UCI

(A ,AeO)20%w/v (O,A,O) NoCI

(am,*) 35% w/v ( . ,A, . ) KCI

.A

( r .O , * ) UBr (O.A.0) NaBr

( . ,A,.) KBr

C

. A

Fig 3. Effect of water activity in equilibrium with the reaction media on the activity of a-chymotrypsin in plastein synthesis, using a peptic hydrolysate of albumin as standard substrate, at three different concentrations (A, 10; B, 20; and C, 35% w/v, respectively). Alkali chlorides and bromides at several concen- trations (0-3 M) were used as water activity depressors in the

reaction medium.

activity was either fully recovered or lost completely. These facts excluded the salts as a a-chymotrypsin specific activators, but the possible deactivated fraction of the enzyme could be involved on the intermolecular associations by hydrophobic interactions with the ' resynthesised' peptide, resulting in the gelling of the plastein reaction mixture, as was previously reported (Edwards and Shipe 1978; Sukan and Andrews 1982).

As a consequence of these facts, the influence of salts on the a-chymotrypsin action in the plastein synthesis must be analysed as a function of the micro- environmental changes during the reaction time : re- duction in water activity of the reaction media, presence of deactivated enzyme and direct substratesalt inter- actions.

Several authors have reported that a substantial reduction in water activity of the medium is needed before the equilibrium of a hydrolytic reaction will be reversed (Hahn-Hagerdal 1986). In this order, the measurements of A , of the reaction media, with or without salts (alkali chlorides and bromides), at various substrate concentrations were made. Figure 3 shows the activity profiles of a-chymotrypsin in the plastein

synthesis against A , of the reaction media. As can be seen, the particular effect of the assayed salts cannot be clearly differentiated, with the exception of the LiBr salt. Additionally, the reduction of A , activity induced by the presence of salts increased slightly the specific activity of the enzyme. This fact establishes clearly that A , was involved in the microenvironmental conditions of the plastein reaction: the main effect of the salts may be on the thermodynamic properties of water and their specific effect seems limited. On the other hand, the reduction in A , was a less important activity-controlling factor compared to the substrate concentration in the plastein synthesis. For example, when a 10% (w/v) substrate concentration was used, the addition of a 3 M salt concentration reduced the A , of 15 %, and induced an increase 3.10-fold in the synthetic activity of a- chymotrypsin; however, when substrate concentration changes from 10 to 20 YO (w/v), the A , was only reduced of 2.30 YO, but the synthetic activity was multiplied by a factor of 3.90. Thus, the increase in the synthetic activity by the substrate concentration was 8.20-fold per unit of A , higher than that produced by the presence of salts.

The effect of deactivated a-chymotrypsin on the plastein synthesis was studied in a reaction model ([S] = 35 YO w/v, [El = 1 O h w/w), using a thermodeactivated a- chymotrypsin solution as additive. Figure 4 shows the plastein production profiles against time as a function of the deactivated enzyme concentration. As can be seen, in all cases, the initial reaction rate remained constant, as a consequence of the non-catalytic power of this additive. However, the final yield of plastein product was increased proportionally to the deactivated enzyme concentration present into the reaction media. This fact clearly shows that the non-specific interaction between the biocatalytic products and the denatured enzyme induces the pro- duction of the water-insoluble and gel-forming products called plasteins.

250 P Lozano, D Combes

0.5

0.4

0.3

E 0.2

c 0 0.1 a3 N w 0.0 0 C 0 0.4

0 v) 0.3

+!

9 0.2

0.1

0.0,

u CI No CI I - t - 0

U B r I mBr

K CI

10 21 24

Elution Volume ( ml )

Fig 5. Gel-permeation chromatograms of the peptic hydro- lysate of albumin used as standard substrate for the plastein reaction in presence of a 2~ alkali chloride or bromide concentration. A FPLC system with a Superose 12@ column and a 50% v/v acetic acid solution as mobile phase at

0.1 ml min-' were used as chromatographic conditions.

Finally, plastein reactions were studied by gel-per- meation chromatography in order to examine the evolution of the profiles against time as a function of the alkali halide present in the reaction medium, using a Superose 12@ (Pharmacia) column. Figure 5 shows the chromatographic profiles of the substrate-peptide mix- ture without enzyme in presence of different salts. As can be seen, the substrate was eluted in several fractions clearly differentiated and, surprisingly, these chromato- graphic profiles were different, as a function of the alkali halide present. For the chlorides the profiles exhibited four fractions, while in the case of the bromides only three fractions were found. Additionally, in both cases, the presence of lithium implied an additional fraction (elution volume 20.5-22 ml). These phenomena deter- mined that strong salt-peptide substrate interactions were developed in the reaction media, which could be interpreted as a modification of the conformational structure of the substrate. The salting-out theory (Melander and Horvath 1977) establishes that the stabilization of a protein in the presence of high salts concentration is determined by the refolding and the reduction of the solubility of hydrophobic groups on the protein molecule, in correlation with the Hofmeister lyotropic series. In the present case, where the activation effect of salts followed these series, this phenomena could be explained by the high exposition of hydrophobic amino acid residues, such as the aromatic residues, of the

substrate by the salting-out effect. Thus, the specificity of the a-chymotrypsin for these residues will imply an enhancement in the biocatalytic action exhibited in the plastein reaction media by the presence of salts.

Finally, the previous study of the plastein reaction by gel-permeation chromatography showed the elution profiles in evolution, as a consequence of the catalytic activity present in the reaction medium (Lozano and Combes 1991). From a general point of view, in all cases, the profiles of the high-molecular-weight fractions were shown to change as a function of time, with an increase in the first and a decrease in the second elution fractions, respectively, corresponding to the synthetic activity present in the reaction medium. Additionally, the latter elution fraction, corresponding to the low-molecular- weight peptides (elution volume 23-26 ml for the bromides, and 22-25 ml for the chlorides), was also increased with reaction time. However, these evolutions of the chromatographic profiles were different as a function of the reaction conditions assayed. Thus, in all cases, the presence of salts in the reaction medium produced an increase in the high-molecular-weight fraction with reaction time higher than in absence of salts. Furthermore, these salts produced an increase in the lowest peptide fraction with reaction time, smaller than in the cases where salts were not present. These facts could be explained by a decrease in the hydrolysis to synthesis product ratio obtained when the substrate concentration was increased. Simultaneously, this de- crease in the low size oligopeptide fraction yield followed the increase in the condensation to transpeptidation product yield ratio, in the presence of salts, as A , reducing agents in the reaction media.

The amino acid composition of all plastein products obtained from the different saline reaction media (3 M salt concentration) and a 35 YO w/v initial substrate concentration, were determined and compared to those of substrate and plastein product obtained from a control reaction without salt (Table 1). The amino acids are classified by the hydrophobicity of their side chain (H4, the molar free energy of transfer for the amino acid with respect to Gly, from an aqueous solution in the organic solvent at the same mole fraction at the limit of infinite dilution), as was described by Bigelow and Channon (1976). The authors have chosen the most hydrophobic amino acids (Hq5 > 1000 cal mo1-l (1 ca z 4.25)) for comparison. In Table 1, the control plastein product showed an overall increase in the hydrophobic amino acid group (55.2% in control plastein versus 44.2 % in substrate). When the alkali halides were present in the reaction media, the content in hydrophobic amino acids was also increased with respect to the control and in the same order as for the activity profiles summarised in Fig 2 (Cl- > Br-; Kf > Na+ > Li'). These results are also in agreement with the salting-out theory of the effect of the Hofmeister lyotropic series to the stability of proteins, where the high exposition of hydrophobic

Alkali halides in plastein reaction 25 1

TABLE 1 Amino acid composition of the different plasteins produced by a-chymotrypsin, using a 35 YO (w/v) peptic hydrolysate of albumin as standard substrate and a 3 M alkali halide concentration into the reaction media, in optimum conditions of assay (40°C; pH 7.0).

Hq5 (cal mol-')

Substrate

Ser Glu ASP GlY Thr Ala His Arg CYS Met LYS Val Leu TYr Phe Pro Ile

Total

- 300 0 0 0

400 500 500 750

1000 1300 1500 1500 1800 2300 2500 2600 2950 -

4.5 1 15.52 9.38 3.09 6.09 8.98 2.53 3.67 2.08 088 9.09 6.69

1220 3.94 6.09 2.89 238

44.16

~

Plastein products

Control LiCl NaCl KCl LiBr NaBr KBr

3.80 4.48 3.0 1 3.30 552 3.87 3.48 8.72 9.53 5.45 4.44 7.42 8.26 8.85 6.46 6.59 3.95 3.63 6.37 5.75 5.90 2.46 2.16 1.69 1.86 3.5 I 2.02 1.87 5.55 5.97 4.93 5.4 1 6.96 5.59 5.76 6.48 6.92 5.98 5.9 1 7.25 6.24 6.41 2.69 3.0 1 2.39 2.65 2.59 2.4 1 2.60 4.37 5.12 454 3.94 4.28 5.04 5.18 4.29 3.53 1.22 1.43 3.0 1 2.96 2.29 1.18 0.44 1.31 0.58 0.58 1.14 1.07 7.97 10.06 7.84 7.19 8.53 8.6 1 9.20 8.94 7.22 9.35 8.18 8.07 8.80 8.86

20.20 19.5 1 27.28 26.74 21.03 23.42 22.68 4.53 3.9 1 6.56 12.2 1 4.20 4.06 3.89 4.8 1 4.73 5.92 5.91 4.50 4.83 4.7 1 2.49 3.74 249 2.07 2.87 3.32 3.43 5.07 3.1 1 5.20 4-55 3.30 3.68 3.82

55.19 5272 66.35 67.43 53.08 57.86 57.66

Below, total hydrophobic amino acids content (Hq5 > 1000 cal mol-') is depicted as a function of the assayed ion.

CI- Bi-

amino acid residues enhanced the plastein synthesis by both the specificity of the enzyme to the aromatic residues and the shifting of the reaction equilibrium by the decrease in the solubility of the reaction products.

CONCLUSIONS

The effect of nine alkali halides (LiF, LiCl, LiBr, NaF, NaC1, NaBr, KF, KCl and KBr) on the plastein reaction catalysed by a-chymotrypsin was studied at different substrate concentrations. The results showed that the presence of salts into the reaction media favoured the plastein production proportionally to their concentra- tion. The analysis of the results allowed us to classify the ions as a function of their activation power (cations : Li+ < Na+ < K+; anions : F- > C1- > Br-), in agreement with the Hofmeister lyotropic series.

In order to know the particular effect of salts on each component of the system (enzyme-substrate-water), the

influence of several factors : A,, presence of deactivated enzyme and salt-substrate interactions, in the plastein reaction was studied. Thus, the A,-depressing power of salts produced a slight increase in the specific activity of the enzyme. The substrate concentration has been shown as the veritable motor in the plastein reaction. Ad- ditionally, the presence of deactivated enzyme increased the final yield of plastein product, but do not affect the initial rate of plastein production. Finally, the structural conformation of the substrate should be changed by the influence of saits, as was shown by the gel-permeation profiles of the substrate.

ACKNOWLEDGEMENTS

The authors are very grateful to Monique Suderie for performing the amino acid analysis. This work was partially supported by the B/BIOT-900024 BRIDGE CEE grant.

252

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