THE INFLUENCE OF SELECTED INHIBITORS ON PROTEOLYTIC ENZYMES ACTIVITY IN FRENCH BEAN

Monika Karaś, Barbara Baraniak
Department of Biochemistry and Food Chemistry, Agricultural University of Lublin, Poland

ABSTRACT

The activity of proteolytic enzymes connected with the albumin fraction of proteins in frozen green French bean cv. Fana as well as the process of inhibition of their activity as result of adding selected chemical compounds (glutathione, Tris, PMSF, EDTA, selected metal ions, diamins, trypsin inhibitor; raw extract of inhibitor isolated from French bean) were determined in the study. Of all the substances tested in the present study silver and mercury ions showed the greatest inhibiting effect on proteases isolated from French bean. Among the organic inhibitors reduced glutathione and PMSF showed the strongest inhibiting activity. Diamins showed the least inhibiting activity (used in the amount ten times higher than in the case of mercury and silver ions they gave a relatively low percentage of inhibition). The protease inhibitors from French bean isolated were inhibitory to their own proteolytic enzymes (extracted with Tris-HCl buffer, pH = 7), as well as to trypsin and pepsin activity, but they showed activating effect on bromelain, that increased along with increasing the amount of the inhibitor extract.

Key words: French bean, protease inhibitors.

INTRODUCTION

In plants, similar to animal organisms, selective hydrolysis of the peptide bond goes on in the specific place of the protein substrate. These reactions may modify the properties and physiological functions of proteins.

Plant raw materials, animal endocrine glands [10], as well as bacteria (Bacillus, Streptococcus, Clostridium) and mildews (Aspergillus) [17] found a broad use. They are used for synthesis of peptides, for modification of proteins contained in food and for producing a lot of new, improved foods. The conditions of proteolytic enzymes controlled enzymatic hydrolysis of proteins in vitro improving their biological value and their functional properties [17, 18, 26].

Proteinases and peptidases catalyze hydrolysis of proteins and peptides during the process of digestion and they play an important physiological and pathological role. Enzymatic hydrolysis of proteins is regulated in several ways, among others by means of specific inhibitors. Protease inhibitors are widespread in the plant world, mainly in the seeds (they constitute up to 0.1-1% protein content in seeds). Most of them are low-molecular proteins or polypeptides containing in their structure untypical amino acids or carbohydrate components.

Proteinase inhibition is a process commonly taking place in nature. Interactions between proteinase and the inhibitor are part of various physiological (e.g. coagulation of blood, fibrinolysis, phagocytosis), pathological ones (e.g. tumorous diseases, hypertension) and infectious process (e.g. AIDS virus or the invasion of parasites) [3]. The widespread occurrence of protease inhibitors in important edible plants and products obtained from them as well as the fact that the control of proteolysis by inhibitors is so specific, initiated numerous studies of their nutritious, toxicological and therapeutic properties, as well as their significance in plant protection [9].

The seeds and leaves of legumes are a natural source of protease inhibitors [22]. These inhibitors are usually easily soluble in water or in diluted acids and are characterized by a high resistance to hydrolysis. Stability of those compounds results from the rigid spatial structure of the molecule sustained by disulphide bonds. Breaking these bonds results in inactivation of the inhibitor.

Specificity of the inhibitors is determined mainly by the structure of the reactive center. Depending on the spectrum of inhibition monovalent (able to inhibit a definite proteolytic enzyme) and polyvalent (inhibiting a few different proteases) inhibitors are differentiated.

In nature, mixed inhibitors occur most frequently which hinders the formation of the enzyme-substrate complex, and in consequence they retard formation and release of the product. The mixed type of inhibition may also occur when a few substrates take part in enzymatic reactions and the inhibitor will reveal a competitive character for some of them while for the others it will reveal a non-competitive one. Also products of protein degradation can show inhibiting action towards proteases. Compound systems occur in living organisms where a lot of different protein substrates in various concentrations occur side by side.

Inhibitors of proteolytic enzymes may play various biological functions [14, 19]:

• they can regulate the intensity of proteolysis in seed tissues,

• they can be the storage protein in the seed,

• they can partly protect the seeds from the negative effect of proteases of microbiological and animal origin,

• they can regulate activity of the enzymes that are in the same plant tissues.

Proteinase inhibitors present in living cells of some plants fulfill the function of a natural regulator of proteolysis. Presence of these substances in food obtained from plants lowers the diet value of the proteins because of the inactivation effect on the digesting enzymes of the alimentary canal.

Limitation of the proteolytic enzymes activity may be caused by the influence of diamines (cadaverine, spermidine and spermine), the products formed as result of decarboxylation of alkali amino acids released during hydrolysis [25].

The aim of the present study was to investigate the activity of proteolytic enzymes isolated from the French bean and the process of inhibiting their activity.

MATERIALS AND METHODS

Samples and chemicals
The French bean (cv. Fana) was planted in the field in Elizówka near Lublin. Mature green pods were harvest and stored at – 20°C.

Proteolytic enzymes (trypsin EC.3.4.21.4-1,880 units/mg solid; pepsin EC.3.4.23.1-515 units/mg solid; bromelain EC.3.4.22.32-2,4 units/mg solid), asoalbumine, reduced glutathione, Tris (hydroxymethyloaminomethane), PMSF (phenylmethanesulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), diamines (spermidine, spermine and cadaverine), trypsin inhibitor, TCA (trichloroacetic acid) were purchased from Sigma (St.Louis, Mo, USA). All other chemicals used were of analytical grade.

Analysis
Extraction French bean protease activity
The albumin fraction with protease activity was extracted from homogenized material with the Tris-HCl buffer pH 7.5. The solid fraction was separated by centrifuging (4000 rpm, for 15 min). In the obtained extracts the proteolytic enzymes activity was determined.

Extraction French bean protease inhibitors
French bean protease inhibitors were extracted from homogenized material with 0.005N HCl. The solid fraction was separated by centrifuging (4000 rpm, for 15 min), then 30% TCA was added to the filtrate and it was again centrifuged (4000 rpm, for 15 min). The obtained filtrate was brought to pH = 8 with 2M NaOH. The obtained extract was a solution of inhibitors.

Inhibiting activity of French bean extracts was tested to trypsin (in the amount 0.2 mg, activity-1,880 units / mg solid), pepsin (0.1 mg, activity-515 units / mg solid), bromelain (0.1 mg, activity-2,4 units / mg solid), as well as proteolytic enzymes (isolated from French bean).

Assay of proteolytic activity
The proteolytic enzymes activity was determined against asoalbumin as substrate [27]. 1 ml of 0.2% asoalbumine solution and 1 ml of the buffer pH 7.5 were added to 1 ml of the extract of the proteolytic enzymes, and incubated for 4 h in the temperature of 310 °K. After that time the reaction was inhibited with 12% TCA and the content was centrifuged (4000 rpm, 15 min). Next 1 ml of 0.6M NaOH was added to the filtrate and absorbance was measured at the wave length λ = 400nm. At the same time a control was carried out by inhibiting the reaction before incubation.

Assay of inhibitory activity
1 ml of 0.2% asoalbumin solution and a varying concentration of the inhibitor were added to 1 ml of the extract of the protease activity isolated from the the French bean and complemented with the Tris-HCl buffer with pH 7.5 to the final capacity 3 ml. Next the samples were incubated for 4 hours at the temperature 37 °C. After this time the reaction was inhibited with 12% TCA and the samples were centrifuged (4000 rpm, 15 min); next 1 ml of 0.6M NaOH was added to the filtrate and the absorbance was measured at the wave length λ = 400nm. No inhibitor was added to the control, in which proteolytic activity of the extracts was measured against asoalbumin.

Next the following inhibitors were tested: reduced glutathione, Tris (hydroxymethylo-aminomethane), PMSF (phenylmethanesulfonyl fluoride), EDTA (ethylenediaminetetraacetic acid), silver and mercury ions – in the amount from 0.02 to 0.1 mM; diamines: spermidine, spermine and cadaverine, magnesium ions – (in the amount from 0.2 to 1 mM; trypsin inhibitor – (in the amount 4, 8, 12, 16, 20 mg); the extract of the inhibitor isolated from the French bean (amounts from 0.1 to 1 ml).

Activity of proteolytic enzymes was expressed in units of activity per g of the sample, accepting increase of absorbance by 0.1 in 4 h as a unit of activity.

The inhibiting activity was expressed as the percentage of inhibition (I) according to the equation: I (%) = [( T - T* ) / T] x 100

where T and T* are the protease activity, without the inhibitor and in its presence respectively.

RESULTS AND DISCUSSION

Investigation of the process of inhibiting the proteolytic enzymes activity isolated from French bean by different inhibitors
Proteolytic enzymes occurring in various parts of plants fulfil many important functions. Action of proteolytic enzymes may be limited by both their protein inhibitors and by compounds with different, non-protein structure. Activity of proteolytic enzymes in the extract from the French beans was 2.19 units/g. All the tested inhibitors caused a decrease of proteolytic enzymes activity. The character of the changes depended on the kind and concentration of the inhibitor. Of all the substances tested in the present study mercury and silver ions showed the most inhibiting effect on proteases isolated from French bean (Fig. 1). Application of 0.02 mM of mercury ions caused a 54% inhibition, and of silver ions – a 33% inhibition. This proves that in the extract there are enzymes whose activity is conditioned by free thiol groups (SH), as even low concentrations of heavy metals ions inactivate this type of enzymes by joining thiol groups. Mercury ions efficiently – in 85% inhibited cysteine proteinases isolated and clarified in a five-stage process (by the use of gel column chromatography, ion-exchange chromatography, and covalent gel chromatography on various carriers) from bean leaves [22]. In the quoted studies cysteine did not show either positive or negative effect on enzyme activity. Among organic inhibitors reduced glutathione and PMSF (phenylmethanesulfonyl fluoride) showed the greatest inhibiting activity. Reduced glutathione, donor of thiol groups, showed a 59% inhibition against proteases isolated from the French bean (Fig. 2a, b). The obtained inhibiting effect is the result of the interaction with proteases present in the extract, whose activity is determined by the presence of other active groups in the catalytic center. In turn, Dionysius et al. [6] found a very low inhibiting activity of glutathione (7%) at the concentration 1 mM against liver-pancreas proteases of crabs (Portunus pelagicus). The obtained differences may result from different origins of the proteases as well as from the fact that the quoted authors used different substrates: TAME (ethyl ester of N-benzoilo-L-arginine) [6], peptides modified by phenol compounds (Z-Phe-Arg-MCA and Z-Arg-Arg-MCA) [22], and in the present study asoalbumine was the substrate of French bean proteases’ action. Both in the extract of crabs’ proteases and in the extract from the French bean analysed in the present study, serine proteases were present. This is proven by the results of inhibition of their activity by PMSF (phenylmethanesulfonyl fluoride). This specific inhibitor of serine proteases (containing the hydroxyl group of serine in its active center and histidine as the donor of the positive charge) revealed a 49% inhibition against proteases from the crab [6]. On the other hand, for serine proteases isolated from tropical Brazilian fish (Brycon orbignyanus) a 26% inhibition was found [10]. PMSF added in the amount of 2 mM inhibited in about 50% the activity of proteinases from langostilla (Pleuroncodes planipes) and from crayfish (Pacifastacus astacus) determined against asocaseine as the substrate in the studies carried out by García-Carreño and Haard [11], and in the concentration of 10 mM for proteases from oat leaves, isolated and clarified by means of various types of column chromatography techniques it showed a 44% inhibition [29]. For endoproteinases isolated from potatoes and separated by two-stage gel chromatography it did not reveal any inhibiting effect [15]. In the present study at the highest tested concentration of PMSF (0.1 mM) a 65% inhibition was found for proteases extracted from the French bean, which proves that there is a significant amount of serine enzymes in them (Fig. 2a, b). This is confirmed by a 42% inhibition obtained after trypsin inhibitor was applied (Fig. 2a, b). This effect is lower than the results obtained by Baraniak and Krzepiłko [2]. In the studies of the process of inhibition of proteases isolated from the seeds of green pea, var. Telefon the quoted authors obtained a 51% of inhibition.

In the case of applying a chelating inhibitor, that is EDTA (ethylenediaminetetraacetic acid) a 47% of inhibition was obtained (Fig. 2a, b). This inhibitor, added in the amount of 5 mM, inhibited in 25% proteases isolated from oat leaves [29], at the concentration of 1 mM inhibited in 13% proteases from potatoes [15], and in the same amount in 10% and 32% proteases isolated, respectively, from langostilla and crayfish [11], but only in 8% proteases from crabs [6]. For serine proteases isolated from tropical Brazilian fish (Brycon orbignyanus) only a 29% inhibition was obtained [10]. The significant inhibiting effect caused by EDTA that was obtained in the present study proves that metaloproteases are present in extracts from the French beans.

Activity of French bean proteases was inhibited by magnesium ions – with the highest tested concentration – by 47% (Fig. 1). Magnesium ions increased the activity of the cysteine proteases fraction, not bonded to Tiopropylo-Sepharose 6B by 5%. The fraction was obtained at the last stage of the process of clarifying the extract from bean leaves. On the other hand, they decreased by 3% this activity in the fraction bonded and eluted with cysteine solution. Magnesium ions also increased by a few percent the activity of proteases isolated from crabs [6]. In the study also the effect of diamines on French bean proteases was investigated. Diamines revealed the weakest inhibiting activity; used in the amount ten times greater than in the case of mercury and silver ions they gave a relatively small inhibition percentage, just a little greater for spermine (Fig. 3). Addition of 0.2 mM of spermine caused a 41% inhibition, of cadaverine – a 34%, and of spermidine – only a 7% inhibition. Polyamines may take part in protein synthesis as regulators [1, 16]. Probably they are indispensable components of cytoplasmatic membranes and cell walls, as any agitation of growth with phytohormones (gibberellins, auxins or cytokinins) induces formation of amines [5]. Presence of polyamines prevents plant organs from aging – they delay degradation of cytoplasmatic membranes (it is suggested that this is done by scavenging free radicals – especially peroxides) [7] and decline of chlorophyll. Considerable amounts of diamines and polyamines are stored in corn grain as well as in broad bean and bean [28]. Polyamines influence enzyme activity by, among others, their covalent bond with enzymatic proteins. As polyamines fulfil various functions in plants, their inhibition effect on proteases isolated from beans was studied. The results obtained in the study that concern the effect of spermine and spermidine (the amines containing the –NH₂ as well as the –NH groups) on proteases isolated from beans point to formation of additional electrovalent bonds (Fig. 3). This fact is also confirmed by the efficiency of inhibition of French bean proteases activity by cadaverine. The bonds lead to a change of the enzymatic protein structure, which ultimately limits their activity.

Investigation of the process of inhibiting the selected proteolytic enzymes activity by inhibitors isolated from French bean
In the seeds of legumes, like in other plants, protease inhibitors commonly occur. The compounds play an important physiological role – that of storage proteins; they also regulate proteolysis intensity and protect the plants from the negative effect of microbiological or animal proteinases [24, 5, 4]. As far as human diet is concerned tripsine inhibitors are seen as compounds that decrease the biological value of food. However, the studies that have been conducted recently show that proteinase inhibitors may have a positive effect on the human organism: anti-cancerogenic [3], anti-fibrinolytic, antiphlogistic; and they may also regulate fats absorption [24]. In the present studies inhibitors isolated from the French bean revealed in 26% the inhibiting activity towards their own proteolytic enzymes (isolated with the Tris-HCl buffer with pH = 7.5) (Fig. 4). Studies conducted by Osman et al. [21] proved the effect of the medium on the activity of trypsine and chymotrypsine isolated from the seeds of bean traditionally consumed in the south-east regions of America. They showed the greatest stability in the acidic medium both when they were present in the raw extract and when they had been purified by chromatographic techniques. This is probably the result of the fast break of the disulfide bonds giving stability and thermal resistance to inhibitor particles that takes place in the alkaline medium. In the study the activity was tested of inhibitors isolated from French bean by the extracting system hydrochloric acid – TCA, towards commercial proteases – pepsine, trypsine and bromelaine (Fig. 5). The extracts inhibited in 28% the pepsine activity and in 50% that of trypsine. Nti and Plahar [20] studied the process of activity inhibition of human and bovine trypsine and chymotrypsine by extracts from seeds of the red variety of cowpea (Vigna unguiculata) subjected to a variety of technological operations. The inhibitors were more efficient towards bovine than human trypsine, however, they drastically inhibited a hydrolytic system containing human chymotrypsine. The studies by Puovreau et al. [23] showed a lower activity of the trypsine inhibitor isolated from pea than that of the commercial bovine inhibitor (Sigma), towards genetically modified forms of trypsine. García-Carreño et al. [12, 13] tested the activity of inhibitors of proteases extracted from seeds of a variety of plants towards proteases isolated from the digestive system of shrimps and proteases isolated from fish muscles. Inhibitors extracted from soya proved to be the most efficient; and proteases from white shrimps were inhibited to a greater degree than proteases from brown shrimps. The inhibitors present in soya beans were also very active towards proteases isolated from fish muscles. They completely inhibited the activity of trypsine, in 80% that of chymotrypsine and in 10% of papaine. The results obtained in the present study, as well as the ones obtained by the quoted authors, may be proof of the presence in plant extracts of inhibitors with a polyvalent character or inhibitors with various specific kinds of activity that is determined by different structures of the active center. Ferrason et al. [8] isolated from peas as many as six protease inhibitors whose molecular masses and the sequence of twenty N-terminal amino acids indicate that they are trypsine inhibitors of the Bowman-Birk type. The increase of inhibition degree resulting from increasing the amount of the inhibitor obtained in the present study proves their competitive or a-competitive action. Protease inhibitors extracted from the French bean did not inhibit bromelaine activity (Fig. 5). They showed activating effect towards this enzyme, which increased along with increasing the amount of the extract. A similar effect of increasing the activity of the plant enzyme – papaine – by extracts from seeds containing trypsine and chymotrypsine inhibitors was obtained in their studies by García-Carreño et al. [13]. Bromelaine and papaine are thiol proteases sensitive to oxidizing factors. In the process of isolating trypsine and chymotrypsine inhibitors from French bean, also substances with a specific kind of action towards their own natural proteases were extracted (Fig. 4). In the case of inhibiting the activity of natural proteases a mixed character of the inhibition process cannot be excluded, as in this extract a few substrates may be present. In such a case the same inhibitor may reveal a competitive action towards one component, it may be an a-competitive inhibitor for another one, and finally protease activity towards one substrate may be inhibited competitively by another substrate. These complicated and ambiguous interactions of active compounds isolated from plants are confirmed by the results of the present study and of the quoted papers.

CONCLUSIONS

The rate of proteases inhibition increases along with the highest concentration of inhibitors. The greatest inhibiting effect showed silver and mercury ions, reduced glutathione and PMSF(phenylmethanesulfonyl fluoride). The inhibitor isolated from the French bean shows greater activity to trypsin, but it showed activating effect on bromelain. The results obtained of the study prove competitive or a-competitive action of the tested inhibitors. Activity, character and efficiency of numerous components are determined by many factors – starting with the species of the plant and methods used in isolating and purifying the compounds, and ending with the precision of the used analytical techniques.

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Accepted for print: 29.11.2006

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