The aim of this piece of work was to assay if the melanins synthesized from  Agaricus bisporus polyphenol oxidase and one of its main substrates (L-tyrosine) act as trypsininhibitors, and therefore may have a toxic effect on protein digestion. It was found that these polymers decrease apparent affinity between trypsin and its substrate (N a-benzoyl-L-arginine ethyl ester, BAEE). In addition, the maximum reaction rate ( rmax) decreases with the increase of melanin concentration (up to 32.6% adding 0.07 mg·mL ‑1).It can be concluded that the presence of melanins fromL‑tyrosine inhibits trypsin activity in a mixed way. Alpha (a)constant was found to be 2.95

Falguera, Víctor

Ibarz, Albert

Revistes Catalanes amb Accés Obert

AfinidAd LXViii, 556, Noviembre - Diciembre 2012 435
RESUMEN
El objetivo de este trabajo fue evaluar si las melaninas sin-
tetizadas a partir de polifenol oxidasa de Agaricus bispo-
rus y uno de sus principales sustratos (L-tirosina) actúan 
como inhibidores de la enzima pancreática tripsina y, por 
tanto, pueden tener un efecto tóxico en la digestión protei-
ca. Se encontró que estos polímeros disminuyen la afini-
dad aparente entre la enzima y su sustrato (Nα-benzoyl-L-
Arginine ethyl ester, BAEE). Además, la velocidad máxima 
de reacción (rmax) disminuye con el incremento de la con-
centración de melanina (hasta un 32,6% añadiendo 0,07 
mg·mL-1). Se puede concluir que la presencia de melani-
nas inhibe la actividad de la tripsina de forma mixta. La 
constante alpha (α) resultó ser 2,95.
Palabras clave: pardeamiento enzimático; inhibición enzi-
mática, digestión, tripsina, melaninas.
SUMMARY
The aim of this piece of work was to assay if the melanins 
synthesized from Agaricus bisporus polyphenol oxidase 
and one of its main substrates (L-tyrosine) act as trypsin 
inhibitors, and therefore may have a toxic effect on protein 
digestion. It was found that these polymers decrease ap-
parent affinity between trypsin and its substrate (Nα-ben-
zoyl-L-arginine ethyl ester, BAEE). In addition, the maxi-
mum reaction rate (rmax) decreases with the increase of 
melanin concentration (up to 32.6% adding 0.07 mg·mL-1). 
It can be concluded that the presence of melanins from 
L-tyrosine inhibits trypsin activity in a mixed way. Alpha (α) 
constant was found to be 2.95.
Keywords: enzymatic browning; enzyme inhibition; diges-
tion; trypsin; melanins.
RESUM
L’objectiu d’aquest treball fou avaluar si les melanines sin-
tetitzades a partir de polifenol oxidasa d’Agaricus bispo-
rus i un dels seus principals substrats (L-tirosina) actuen 
com a inhibidors de l’enzim pancreàtic tripsina i, per tant, 
poden tenir un efecte tòxic en la digestió proteica. Es va 
trobar que aquests polímers disminueixen l’afinitat aparent 
entre l’enzim i el seu substrat (Nα-benzoyl-L-arginine ethyl 
ester, BAEE). A més a més, la velocitat màxima de reac-
ció (rmax) disminueix amb l’increment de la concentració de 
melanina (fins a un 32,6% afegint-ne 0,07 mg·mL-1). Es pot 
concloure que la presència de melanines inhibeix l’activi-
tat de la tripsina de manera mixta. La constant alpha (α) 
resultà 2,95.
Paraules clau: embruniment enzimàtic; inhibició enzimàti-
ca; digestió; tripsina; melanines.
Inhibitory effect of enzymatic browning 
products on trypsin activity
Víctor Falguera1 and Albert Ibarz
Departament de Tecnologia d’Aliments UPV-XaRTA, (Universitat de Lleida), Av. Rovira Roure, 191, 25198 Lleida (Spain)
Efecto inhibitorio de los productos del pardeamiento enzimático sobre la actividad de la tripsina
Efecte inhibitori dels productes d’embruniment enzimàtic sobre l’activitat de la tripsina
Recibido: 24 de octubre de 2011; aceptado: 12 de enero de 2012
1Corresponding author: vfalguera@tecal.udl.cat; 
Tel.: (34) 973 702521; Fax.: (34) 973 702596
AfinidAd LXViii, 556, Noviembre - Diciembre 2012436
1.INTRODUCTION
Enzymatic browning products are a heterogeneous group 
of polymers formed by polyphenol oxidase action in veg-
etable tissues containing phenolic or polyphenolic mol-
ecules, which have been considered up to the present 
substances that produce deterioration in many foods, es-
pecially in mushrooms, fruit juices and other fruit deriva-
tives, decreasing its market value. Nevertheless, recent 
discoveries on beneficial properties on health, such as 
anti-oxidative, anti-inflammatory, immune and anti-tumour 
properties, have done that not only their elimination should 
be reconsidered, but also their addition could be proposed 
in order to take advantage of these properties (Falguera et 
al., 2010a).
Polyphenol oxidase (E.C. 1.14.18.1) is a copper-contain-
ing enzyme that catalyzes two distinct reactions involving 
molecular oxygen with various phenolic substrates: the 
o-hydroxylation of monophenols to o-diphenols (cresolase 
activity) and the subsequent oxidation of o-diphenols to 
o-quinones (catecholase activity). The later polymerization 
of these compounds leads to the formation of the enzy-
matic browning final products, called melanins (De Faria et 
al., 2007; Falguera et al., 2010a).
It is accepted that processing and storing of foodstuffs 
may cause a reduction in the quality of the constituents, 
affecting both its biological value and its digestibility (Öste 
& Sjödin, 1984). In some cases, protein digestibility may 
be affected by the formation of non-enzymatic browning 
(NEB) products, as it has been reported by several authors 
(Öste & Sjödin, 1984; Öste et al., 1986; Hirano et al., 1994; 
Ibarz et al., 2008; Ibarz et al., 2009). Thus, protease in-
hibitors have been recognized to be significant in protein 
metabolism and endocrine systems. More than 100 kinds 
of proteinous and non-proteinous inhibitors have been 
isolated and identified from various living bodies such as 
animals, plants and microbes (Hirano et al., 1994).
Trypsin (E.C. 3.4.21.4), as pepsin and chymotrypsin, is 
one of the main digestive proteases. It is synthesized in 
the pancreas in the inactive form of trypsinogen, and its 
activity is located in the small intestine where it degrades 
proteins to polypeptides and amino acids in a medium pH 
of about 8.0 (Ibarz et al., 2009). Concretely, it catalyses 
the hydrolysis of the peptidic and ester bonds formed by 
the carboxyl group and the basic amino acids L-lysine and 
L-arginine.
Trypsin activity is negatively influenced by physical param-
eters (temperature and pH), by conformational changes 
(denaturalization), by chemical modifications (substitution 
of amino acid residues and reduction of disulphite bridg-
es) or by specific interactions with inhibitors. Sometimes, 
the formation of the catalytically inactive enzyme-inhibitor 
complex can be useful to understand the formation of the 
enzyme-substrate complex and the interactions that occur 
during the catalysis (Schellenberger et al., 1994).
Thus, on the one hand, melanins have been reported to be 
beneficial for health, but on the other hand it has been ob-
served that similar polymers (NEB products) have a toxic 
effect on proteases activity. The aim of this work has been 
to assay if enzymatic browning products synthesized by 
means of Agaricus bisporus polyphenol oxidase act as 
trypsin inhibitors and, in that case, to determine the kind 
of inhibition and its kinetic parameters.
2. MATERIALS AND METHODS
2.1. Melanin preparation
Agaricus bisporus polyphenol oxidase (Sigma Chemical, 
St. Louis, MO) was diluted in a 50 mM sodium phosphate 
buffer (pH 6.5) to an enzyme solution activity of 500 U·mL-1, 
distributed in aliquots of 1 mL and frozen at -12ºC until 
use. This solution was stored at 4ºC since 12 hours before 
experiments started, and then pre-incubated at room tem-
perature (22±2ºC) for 1 hour. L-Tyrosine (Sigma Chemical, 
St. Louis, MO) was prepared in sodium phosphate buffer 
(pH 6.0, following the optimal pH determined in a previous 
piece of work (Falguera et al., 2010b)) in a concentration of 
4.0 mM. The final enzyme content in the reaction mixture 
was 10 U/mL. 
After 24 h of reaction, melanins were precipitated adjusting 
the solution pH to 2.0 with HCl. The mixture was centri-
fuged in an Avanti J-26XP Centrifuge (Beckman Coulter, 
USA) for 12 minutes at 12,000 rpm. The supernatant was 
discarded and the pellet was recovered with distilled wa-
ter. Melanins were lyophilized and rediluted in dimethyl sul-
foxide (DMSO) in a concentration of 0.82 mg/mL.
2.2. Trypsin activity determination
The principle of the enzymatic assayed reaction is the ac-
tion of bovine trypsin (Sigma Chemical, St. Louis, MO) on 
Nα-benzoyl-L-arginine ethyl ester (BAEE) in aqueous so-
lution at pH 7.6 and 25 °C, giving Nα-benzoyl-L-arginine 
(Bz-L-Arg) and ethanol (Bergmeyer et al., 1974). This reac-
tion can be followed measuring the solution absorbance 
at 253 nm with a 1 cm width quartz cell, using an Helios 
gamma spectrophotometer (Thermo Fisher Scientific Inc., 
Waltham, USA) and monitoring the results with the Vision 
Lite 1.0 software (Thermo Spectronic, Thermo Fisher Sci-
entific Inc., Waltham, USA). A data point was taken every 3 
s. Trypsin activity was expressed as the absorbance vari-
ation (formation of Bz-L-Arg) for each minute and protein 
amount of the enzyme (DA
253 s
-1 mg-1). 
2.3. Trypsin inhibition analysis
Experimental series were carried out in a substrate con-
centration range from 0.2 to 0.6 mM, since higher BAEE 
concentrations led to inhibition by substrate (Ibarz et al., 
2009). A trypsin standard solution containing 500 U/mL 
and three melanin solutions with concentrations of 0.82, 
0.41 and 0.205 mg/mL in DMSO were prepared.
The reaction mixture contained 3 mL of substrate and 300 
mL of a melanin solution (final melanin contents: 0.07, 
0.035 and 0.0175 mg/mL). 20 mL of the enzymatic solution 
(10 U) were added to the different mixtures (final enzyme 
content: 0.22 mg/mL) and the absorbance at 253 nm evo-
lution was monitored. Blank experiments were carried out 
in order to prove that there was not any interference of the 
different substances (mainly melanin and DMSO) with the 
absorbance at 253 nm.
2.4. Data processing and statistical analysis
From the increase of the absorbance with the time of reac-
tion, it is possible to obtain the maximum reaction rates 
for each substrate and melanin concentration, which is 
reached at zero time. With this aim, the monitored varia-
tion in the absorbance at 253 nm with reaction time can be 
fitted to exponential curves:
              (1)
AfinidAd LXViii, 556, Noviembre - Diciembre 2012 437
From this expression it is possible to obtain the initial reac-
tion rate, as that is the value of its derivative at the initial 
time (Ibarz et al., 2008):
                (2)
From the data of the initial rate of reaction for the different 
substrate and melanin values used, the Lineweaver–Burk 
method allows to obtain the Michaelis-Menten (MM) kinet-
ic type parameters, which are the MM constant (KM) and 
the maximum reaction rate (rmax) (Segel, 1982). The data 
from this representation was adjusted to a straight line by 
means of the least squares method.
The fittings to the different kinetic and mathematical ex-
pressions were carried out using Statgraphics Plus 5.1 
statistical data processing software (STSC Inc., Rockville, 
MD, USA). The fittings and the estimates were calculated 
at a 95 % significance level. All shown results are the aver-
age of three determinations.
3. RESULTS AND DISCUSSION
In order to study the effect of the melanin synthesized from 
L-tyrosine on trypsin activity, three different melanin con-
centrations were assayed on BAEE solutions. It was ob-
served that the initial reaction rate had a great dependence 
on melanin concentration, being lower as it increased. To 
characterize and quantify this inhibitory effect these initial 
reaction rates were transformed following the Lineweaver-
Burk method (Figure 1). 
figure 1. Lineweaver-Burk representation of samples with 
different concentrations of melanin from L-tyrosine.
Table 1. Kinetic parameters for trypsin in-
hibition by melanin from L-tyrosine.
CMelanin (mg/mL) KM (mM) rmax (DA253·s
-1·mg-1) R2
0 0.86 ± 0.16 0.0108 ± 0.0027 0.9345
0.0175 0.96 ± 0.44 0.00990 ± 0.00039 0.9987
0.035 1.09 ± 0.13 0.00979 ± 0.00095 0.9938
0.07 1.10 ± 0.19 0.0092 ± 0.0015 0.9526
Table 1 shows the values of MM constant and maximum 
reaction rates for the experimental series carried out with 
different melanin concentrations. MM constant increases 
with the melanin concentration in the solution, which in-
dicates that this polymer decreases the apparent affinity 
between trypsin and its substrate (Nelson & Cox, 2000). 
In addition, the maximum reaction rate tends to decrease 
with the increase of melanin content in a linear tendency. 
The highest obtained inactivation was 32.6% with a BAEE 
concentration of 0.6 mM and a melanin content of 0.07 
mg·mL-1. By extrapolation of this linear tendency, the nec-
essary melanin concentration to inhibit the enzyme com-
pletely would be 0.50 mg·mL-1. However, this deduction 
could not be empirically proved, since melanin solubility 
made it impossible to work with concentrations higher 
than 0.25 mg·mL-1. At higher melanin contents, some of 
the added polymer remained constantly insoluble.
These facts are evidence enough to determine that the 
presence of melanin from L-tyrosine inhibits trypsin activ-
ity in a mixed way (Segel, 1982). Inhibition ways are rep-
resented in Figure 2. Since melanins are a heterogeneous 
group of polymers with different chain lengths, the differ-
ent fractions are expected to act in different moments of 
the catalysis, joining both trypsin alone or trypsin-BAEE 
complex and leading to mixed inhibition kinetics. Ibarz et 
al. (2009) found a similar behavior in the interaction be-
tween trypsin and melanoidins, which is also a heteroge-
neous group of polymers with different chain lengths.
figure 2. Mixed enzymatic inhibition mechanism (adapted 
from Segel, 1982). T: trypsin. BAEE: Nα-benzoyl-L-arginine 
ethyl ester. Bz-L-Arg: Nα-benzoyl-L-arginine. EtOH: ethanol 
According to Jencks (1969), the electrostatic and apo-
lar contacts dictate enzyme-substrate complementarity, 
which is necessary for surmounting the activation ener-
gy barrier between the ground and transition states. As 
substrate binding sites are preformed and relatively rigid, 
the free energy of substrate binding can be converted to 
catalysis without a large entropic penalty. Therefore, rate 
acceleration must also depend on the ability of distal por-
tions to stabilize the binding, so the whole protein archi-
tecture must play an important role (Perona et al., 1995). 
Moreover, it is known that the differences in substrate 
specificity between trypsin and chymotrypsin are provided 
by variations in the distal portions that create a particular 
electrostatic environment, since the structure of the active 
site is the same in both enzymes (Stroud, 1974; Hedstrom 
et al., 1992). Then, any molecule present in the reaction 
medium may have an active effect on these electrostatic 
and apolar bindings, modifying the local environment that 
is necessary to create the enzyme-substrate links. Melanin 
chains with different molecular weight may create bindings 
with different sites of trypsin molecules, either blocking the 
active site or modifying these electrostatic forces. 
The conclusion that the inhibition is mixed-type can also 
be stated observing that the intersection of the regression 
lines in the Lineweaver-Burk plot is found in the second 
quadrant. Thus, the inhibition constant αKi (enzyme-sub-
strate-inhibitor complex) must be higher than the inhibi-
tion constant Ki (enzyme-inhibitor complex), and then the 
value of α will be higher than the unit. To obtain the values 
of these parameters, the slope and the intercept of the 
Lineweaver-Burk regressions were represented in front of 
the inhibitor concentration (Segel, 1982). Ki was found to 
be 0.148 mg·mL-1, while αKi was 0.438 mg·mL
-1. Thus, α 
AfinidAd LXViii, 556, Noviembre - Diciembre 2012438
value was 2.95. The fact that α is higher than the unit, but 
close to it, indicates that, indeed, the inhibition is mixed-
type (Copeland, 2000), supporting the evidence observed 
in Figure 1. Ibarz et al. (2009) found an α value of 1.88 in 
the inhibition of trypsin by melanoidins synthesized from 
glucose and asparagine. 
4. CONCLUSIONS
To sum up, it can be stated that melanins synthesized from 
L-tyrosine and Agaricus bisporus polyphenol oxidase have 
an inhibitory effect on trypsin activity. The higher mela-
nin concentration was, the more important the inhibition 
factor was found. The highest obtained inactivation was 
32.6%, corresponding to a BAEE concentration of 0.6 mM 
and a melanin content of 0.07 mg·mL-1. MM constant (KM) 
increased with the melanin concentration in the solution, 
which indicates that this polymer decreases the apparent 
affinity between trypsin and its substrate. In contrast, the 
maximum reaction rate (rmax) tends to decrease with the in-
crease of melanin content in a lineal tendency. These facts 
show that this inhibition is mixed-type.
ACKNOwLEDGEMENT
V. Falguera: The research leading to this work has been 
partially supported by the Programa de Formación del Pro-
fesorado Universitario from the Ministerio de Educación of 
the Spanish Government.
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Inhibitory effect of enzymatic browning products on trypsin activity

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