Investigation of glutathione peroxidase activity in chicken meat

661

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

Original

Ciência e Tecnologia de Alimentos ISSN 0101-2061

Received 16/3/2010

Accepted 28/2/2012 (004730)

Departament of Technology and Science of Food, Universidade Federal de Santa Maria – UFSM, Bairro Camobi, CEP 97105-900, Santa Maria, RS, Brazil,

e-mail: ajcichoski@hotmail.com

Departament of Food Engineering, Universidade Regional Integrada do Alto Uruguai e das Missões – URI, Campus de Erechim, Av. Sete de Setembro, 1621,

CEP 99700-000, Erechim, RS, Brazil

EMBRAPA Pigs and Poultry, BR 153, Km 110, CP 21, CEP 89700-000, Concórdia, SC, Brazil

*Corresponding author

Investigation of glutathione peroxidase activity in chicken meat under dierent

experimental conditions

Investigação da atividade de glutationa peroxidase em carne de frango submetida a diferentes

condições experimentais

Alexandre José CICHOSKI

*, Renata Bezerra ROTTA

, Gerson SCHEUERMANN

Anildo CUNHA JUNIOR

, Juliano Smanioto BARIN

1 Introduction

Glutathione peroxidase (GSH-Px) is a selenoenzyme that is

able to slow down and prevent oxidative reactions in muscular

tissues by controlling free radicals formation from preexisting

peroxides (ARAIetal., 1994). GSH-Px is naturally found in

bones and inhibits lipid oxidation in both live tissues and

post-slaughter meat (CHAN; DECKER, 1994). Hence, GSH-Px

Resumo

Uma vez que estudos anteriores sobre a atividade enzimática da glutationa peroxidase (GSH-Px) divergem acerca da metodologia e dos

resultados, este estudo teve por objetivo investigar a inuência de diferentes condições de ensaio sobre a atividade da GSH-Px em coxas de

frangos, oriundos de diferentes dietas, nas quais se variaram a fonte e a concentração de selênio. As atividades da GSH-Px foram determinadas

seis horas após o abate e 120 dias após o armazenamento a –18 °C. As diferentes condições de ensaio envolveram a pré-incubação (0,

10 e 30minutos), o meio de reação, os tipos de substratos [H

(0,72 mM, 7,2 mM e 72 mM) e terc-butil hidroperóxido 15 mM] e as

concentrações diferentes de tampões [tampão 1 (fosfato de potássio 50 mM pH 7,0+EDTA 1 mM+mercaptoetanol 1 mM) e tampão

2(tris-HCl 50 mM pH 7,6+EDTA 1 mM+mercaptoetanol 5 mM)] no meio de reação. Os resultados mostraram que: i) a maior atividade

da GSH-Px foi observada quando a enzima e o substrato foram colocados em contato a 22 °C sem qualquer pré-incubação; ii) o peróxido de

hidrogênio, quando utilizado em concentrações acima de 0,72 mM, saturou a enzima GSH-Px e inibiu sua atividade; iii) a enzima GSH-Px

apresentou maior anidade para o substrato peróxido de hidrogênio, quando comparado ao peróxido de terc-butil, e iv) a adição do tampão

mercaptoetanol não promoveu aumento na atividade da enzima GSH-Px. A atividade da GSH-Px também não foi inuenciada pela fonte e

pela concentração de selênio vindo pela dieta. Os resultados obtidos permitiram denir qual a melhor temperatura de contato entre a enzima

e o substrato (22 °C) e qual a melhor concentração e o tipo de substrato e de tampão a serem utilizados. Essas informações poderão servir de

base para a execução de futuros trabalhos envolvendo a determinação da atividade da GSH-Px em carnes, pois há contradições nas poucas

informações existentes na literatura.

Palavras-chave: GSH-Px; carne de frango; atividade enzimática; selênio; tampão; substrato.

Abstract

Due to the fact that previous studies on the enzymatic activity of Glutathione peroxidase (GSH-Px) diverge widely in their methodology and

results, this study aimed to investigate the inuence of dierent analytical conditions on GSH-Px activity in chicken thighs from broilers that

were fed dierent diets with dierent sources and concentrations of selenium. GSH-Px activity was evaluated six hours aer slaughter and

120 days aer frozen storage at –18 °C. e dierent analytical conditions included time of pre-incubation (0, 10 and 30 minutes), reaction

medium, types of substrate (H

(0.72 mM, 7.2 mM, and 72 mM) and Terc-butil hydroperoxide 15 mM), and dierent buer concentrations

(buer 1, potassium phosphate 50 mM pH 7.0+EDTA 1 mM+mercaptoethanol 1 mM, and buer 2, tris-HCl 50 mM pH 7.6+EDTA

1mM+mercapthanol 5 mM). e results show that the highest GSH-Px activity was observed when enzyme and substrate were in contact at

22 °C without any pre-incubation, and that, when used at concentrations above 0.72 mM, hydrogen peroxide saturated the GSH-Px enzyme

and inhibited its activity. e enzyme presented higher anity to hydrogen peroxide when compared to terc-butil peroxide, and the addition

of a buer containing mercaptoethanol did not increase GSH-Px enzymatic activity. e activity of GSH-Px was not inuenced by the source

and concentration of selenium in the diet either. e obtained results allowed the determination of the best temperature of contact between

the enzyme and substrate (22 °C), the optimum concentration, and the type of substrate and buer to be used. is information is extremely

useful for future studies on GSH-Px activity in meat due to the divergence and little information found in the literature.

Keywords: GSH-Px; meat chicken; enzymatic activity; selenium; buers; substrate.

OI:D http://dx.doi.org/10.1590/S0101-20612012005000107

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

662

Investigation of glutathione peroxidase activity in chicken meat

into two stages: the rst stage comprised ve experiments, in

which the thigh meat enzymatic activity was evaluated six

hours aer slaughter; the second stage was carried out on based

on the results obtained in the rst stage. In the second stage,

the material used to measure GSH-Px activity was raw meat

frozen for 120 days at –18 °C. e samples were randomly

chosen among the chickens fed T2 and T5 diets. In both stages,

three replicates were made for each analysis. Means, standard

deviation, analysis of variance, and the Tukey’s test of means

were performed at 5% condence level using Statistica (Statso,

Tulsa, USA) soware, version 6.1.

2.2 GSH-Px determination in chicken thigh meat six hours

aer slaughter

The enzymatic extract was obtained according to the

modied method proposed by Devoreetal. (1983). Skinless and

visually fat-free chicken thighs were homogenized with tris-HCl

buer 50 mM at pH 7.6 and 5°C at the 1:5 w/v ratio. Next, the

mixture was centrifuged (27.500 g, 30 minutes, 4°C), and the

supernatant was ltered and once again centrifuged under the

same conditions.

e GSH-Px activity of enzymatic extract was determined

according to a modication of the method proposed by Paglia

and Valentine (1967). e reaction medium was composed of

potassium phosphate buer 171 mM, sodium azide 4.28 mM,

EDTA 2.14 mM, reduced glutathione 6 mM, NADPH 0.9 mM,

and glutathione reductase 2 U.mL

–1

. e reaction took place

at 22°C (±1), starting with the addition of H

0.72 mM.

e absorbance of the samples was measured at 340nm using

a spectrophotometer. e measurements were taken every

15seconds for 300 seconds. e GSH-Px enzymatic activity

can be expressed in enzymatic units permL of sample (U.mL

–1

U.L

–1

, U.g

–1

of tissue, U.g

–1

of protein, or U.mg

–1

of hemoglobin

(PUNCHARD; KELLY, 1996). In the present study, the GSH-Px

enzymatic activity was expressed as U.g

–1

of tissue and was

calculated using Equation1:

/ / minUg A F

=∆×

(1)

where F is a constant used for converting absorbance per

minute (∆A/min) into enzymatic units (U). F is calculated by

the following Equation2:

( / )5

6.22

RV SV

(2)

where RV is the reaction volume (inmL); SV is the sample

volume (inmL); 5 is the volume (inmL) used to dilute 1 g of

tissue during the enzyme extraction; and 6.22 is the NADPH

molar extinction coecient (in Mm.cm

–1

2.3 GSH-Px determination in chicken thigh aer 120 days of

storage at –18 °C

The enzymatic extract was obtained according to the

same procedure described above, except from the fact that

two dierent buers where tested in the reaction medium:

buer 1, composed of potassium phosphate 50 mM at pH of

activity has been measured in several types of meat, such as

cattle (LEE; MEI; DECKER, 1996a, 1996b; O’GRADY etal.,

2001), pork (MAHAN; PARRETT, 1996; HERNÁNDEZ; PARK;

RHEE, 2002), and poultry (MOREIRAet al., 2001; SURAI,

2002; CARRERASetal., 2004, HOACetal., 2006). GSH-Px

catalyzes the detoxication of hydrogen peroxide (H

) in vitro

presenting high specicity for this substrate (ROTRUCKetal.,

1973). In fact, such reaction takes place in vivo through a parallel

oxidation of the reduced form of glutathione (GSH), a tripeptide.

e resulting molecule, oxidized glutathione (GSSG), is reduced

back through a parallel reaction with the reduced form of

nicotinamide adenine dinucleotide phosphate (NADPH).

is reaction is catalyzed by the glutathione reductase enzyme

(ROVERJUNIORetal., 2001).

Considering these reactions, Paglia and Valentine (1967)

proposed an analytical method for the determination of GSH-Px

enzymatic activity in erythrocytes. According to this method, the

decrease in the NADPH absorbance at 340nm, in the presence

of H

, is proportional to GSH-Px enzymatic activity. Since

its proposal, this has been the standard method for measuring

GSH-Px activity in all kinds of samples due to its convenience

and reliability. (CARRERASetal., 2004; DAUN; AKESSON,

2004a, 2004b; HOACetal., 2006; HOLOVSKÁJUNIORetal.,

2003). Despite the existence of several studies on GSH-Px

activity measurement, the inuence of experimental parameters

for estimating GSH-Px activity is still not clear. e standard

technique is an indirect and complex method, and there are

some uncertainties about the reaction mechanisms involved.

In addition, the studies conducted on this topic diverge widely

in their methodology and results. Due to the little information

available about the analytical method and the divergences

concerning the determination of GSH-Px activity in the

muscle, this study aimed to investigate the inuence of dierent

experimental conditions on the activity of GSH-Px in chicken

thighs. erefore, the type and concentration of peroxide, the

buers, the inuence of reaction time and temperature at the

analytical signal, as well as the inuence of broiler’s diet and

the eect of storage were studied, and the optimized conditions

were established.

2 Material and methods

2.1 Materials

One thousand and one hundred forty male chickens

(lineage Ross) were fed ve dierent diets: T1, without selenium

supplementation; T2, supplemented with inorganic selenium

(sodium selenite) at the concentration of 0.15mg.kg

–1

; T3,

supplemented with inorganic selenium at the concentration of

0.35mg/kg; T4, supplemented with organic selenium (Sel-Plex

)

at the concentration of 0.15mg.kg

–1

; and T5, supplemented

with organic selenium at the concentration of 0.35mg.kg

–1

e diets were elaborated from basic formulations following

recommended nutritional requirements (ROSTAGNO etal.,

2005).

At the age of 42 days, ninety birds were slaughtered, and

their thigh meat was used as raw material for evaluating the

glutathione peroxidase (GSH-Px) activity. e study was divided

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

663

Cichoski et al.

Stagsted (2006) observed the same behavior when measuring

GSH-Px activity in milk using the standard Paglia and Valentine

method and also obtained dierent results from other studies.

erefore, they decided to vary the enzymatic assay conditions

in order to optimize the method.

3.2 GSH-Px activity in chicken thigh aer 120 days of

storage at –18 °C

Eect of dierent hydrogen peroxide concentrations on GSH-Px

activity

e GSH-Px enzyme reduces several reactive oxygen species

(ROS) including hydrogen peroxide (CHAUDIEREetal., 1984).

e rst step in such reduction reaction is a direct oxidation of

selenolate anion (E-Se

–

) or selenol (E-SeH), which are the two

catalytically active forms of the selenocysteine residue contained

in GSH-Px (PRABHAKARetal., 2005). is reaction is shown

in Equation3.

22 2

––

E SeH H O E SeOH H O+→ +

(3)

Aer the oxidation of the GSH-Px active center by hydrogen

peroxide, the enzyme reacts with a reduced glutathione (GSH)

molecule forming an enzyme-glutathione complex (Equation4).

Next, this complex reacts with another GSH molecule, which

reduces back the GSH-Px enzyme and releases an oxidized

glutathione molecule (Equation5). Finally, GSH-Px reduces

once again the oxidized glutathione molecule in the presence

of NADPH (PRABHAKARetal., 2005).

– ––E SeOH GSH E Se SG H O+→ +

(4)

–– –

E Se SG GSH E SeH GSSG

+→ +

(5)

Since hydrogen peroxide is a substrate for the GSH-Px

enzyme, its concentration controls the reaction speed. us,

lack or excess of hydrogen peroxide in the reaction medium

could aect the enzymatic kinetics. In order to evaluate the

eect of hydrogen peroxide concentration on the GSH-Px

reaction rate, three dierent hydrogen peroxide concentrations

were used in the preparation of the reaction medium: 0.72mM,

7.2 mM, and 72 mM. e results shown in Figure1 show

that the reaction rates were signicantly aected by hydrogen

peroxide concentration. According to Lehninger, Nelson and

Cox (1995), all enzymes can be saturated by a proper substrate,

7.0, EDTA 1 mM, and mercaptoethanol 1 mM; and buer 2,

composed of tris-HCl 50 mM at pH of 7.6, EDTA 1 mM and

mercaptoethanol 5 mM. In both cases, the extraction took place

at 5°C with a 1:5w/v ratio. Next, the mixture was centrifuged

as described above and the supernatant was ltered and once

again centrifuged under the same conditions.

e GSH-Px activity of enzymatic extract was determined

according to the same method described above. In this stage,

however, the reaction took place at 36 °C aer a pre-incubation

time of 10 or 30 minutes. In addition, two substrates were tested

under dierent concentrations: H

at the concentrations of

0.72 mM, 7.2 mM, and 72 mM; and terc-butil hydroperoxide

at the concentration of 15 mM. e absorbance was measured

under the same conditions previously described, and GSH-Px

enzymatic activity was expressed in U.g

–1

of tissue and calculated

using Equations1 and2.

3 Results and discussion

3.1 GSH-Px activity in chicken thigh meat six hours aer

slaughter

Table1 shows the GSH-Px activity values in chicken thigh

meat six hours aer slaughter considering the dierent diets of

the birds with inorganic or organic selenium.

e results show that changing the selenium source and

concentration in the diet did not inuence GSH-Px activity

in chicken thighs. HolovskáJunioretal. (2003) studied the

eect of four diets (without selenium, 0.2mg of inorganic

selenium.kg

–1

, 0.2mg of organic selenium.kg

–1

, and 0.3mg

of organic selenium.kg

–1

) on chicken liver GSH-Px activity.

ey observed that changing the birds diet did not inuence

the chicken liver GSH-Px activity. A study conducted by Daun

and Akesson (2004a) reports similar results: the dierences

in GSH-Px activity in chicken thighs were not related to the

levels of selenium in the diets. Such results could be attributed

to the fact that the selenium ingested by the birds is used

for producing several selenoproteins besides GSH-Px. us,

selenium distribution in the bird body is regulated by its

metabolic needs (DAUN; AKESSON, 2004b). For example, birds

subjected to heat or cold stress present an increase in the levels

of the selenoprotein Type I Iodothyronine Deiodinase, which

regulates body temperature (ARTHUR; NICOL; BECKETT,

1993). Since thirty selenoproteins have been previously reported,

it is quite possible that the selenium added to the birds diet in

the present study was used for other metabolic activities than

the GSH-Px synthesis. e GSH-Px activity values observed in

our study were similar to those observed by HOACetal. (2006)

in chicken breast (0.050 U.g

–1

) but lower than those observed by

Daun and Akesson (2004a) in chicken breast (0.7 to 1.0 U.g

–1

According to Punchard and Kelly (1996), the ideal decay

rate of absorbance for the GSH-Px enzyme is between 0.01 and

0.05 U/min. In the rst stage of the present study, the absorbance

decay rate was much lower than these values. erefore, it was

necessary to study the inuence of changing the enzymatic

assay conditions on GSH-Px activity in order to optimize the

method and also study details of the GSH-Px enzymatic kinetics.

Table 1. Inuence of dierent diets (T1, T2, T3, T4, and T5) on

GSH-Px enzymatic activity (U.g

–1

) in chicken thigh meat six hours aer

slaughter. e data are expressed as mean ± standard deviation (n = 3).

Treatment Enzymatic activity (U.g

–1

tissue)

T1 (no Se) 0.051

(± 0.031)

T2 (inorganic Se at 0.15mg.kg

–1

) 0.083

(± 0.14)

T3 (inorganic Se at 0.35mg.kg

–1

) 0.045

(±0.003)

T4 (organic Se at 0.15mg.kg

–1

) 0.077

(± 0.015)

T5 (organic Se at 0.35mg.kg

–1

) 0.085

(± 0.019)

Dierent letters in the same column indicates signicant dierence by the Tukey test

(p < 0.05).

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

664

Investigation of glutathione peroxidase activity in chicken meat

during the pre-incubation period in water bath. erefore,

when the absorbance readings were made, the NADPH was

almost fully oxidized yielding reaction rates closer to zero. is

behavior was expected, since the reactions catalyzed by cellular

GSH-Px enzyme, found in the muscles, are faster than those

catalyzed by extracellular enzymes, found in milk and blood

plasma (STAGSTED, 2006). ose two enzyme subspecies

are structurally and functionally dierent from each other.

Extracellular GSH-Px enzyme presents a higher resistance to

high temperatures when compared to cellular GSH-Px enzyme

(LINDMARK-MANSSONetal., 2001). erefore, GSH-Px

might have been inhibited by the temperatures used for pre-

incubation in the present study.

Eect of dierent substrate types on the GSH-Px activity

Hydrogen peroxide and terc-butil hydroperoxide are

substrates for GSH-Px enzyme presenting Michaelis-Menten

constants (K

) of 0.003 and 0.059 mM, respectively (BRENDA,

but the substrate concentration required to saturate each one of

them is variable. In this study, hydrogen peroxide concentrations

above 0.72 mM reduced the reaction rate, i.e., saturating the

enzyme and inhibiting its catalytic activity. Splittgerber and

Tappel (1979) used three types of hydroperoxides at dierent

concentrations as substrates for mice liver GSH and GSH-Px

keeping the enzyme concentration constant. ey observed that

an increase in hydroperoxides concentration caused a decrease

in the reaction rates. Lin and Hultin (1978) also reported that

the GSH-Px enzyme is easily saturated by hydrogen peroxide.

erefore, high concentrations of hydrogen peroxide reduce the

reaction rate by slowing down the initial reaction (Equation3).

Eect of dierent pre-incubation conditions of the reaction

medium on the GSH-Px activity

According to previous ndings, the GSH-Px enzymatic

activity is aected by the pre-incubation time and temperature

of the reaction medium. In the study of Carrerasetal. (2004), the

reaction medium was pre-incubated at 30°C for 5 minutes prior

to the addition of hydroperoxide and absorbance readings. On

the other hand, Moreiraetal. (2001) and Penha-Silvaetal. (2005)

pre-incubated the medium at 37°C for 10 minutes before adding

the starter. Some authors report that the immediate reaction

takes place at 37°C, without pre-incubation (LINDMARK-

MANSSONetal., 2001; HOLOVSKÁJUNIORetal., 2003;

HOACetal., 2006). Other studies report the use of temperatures

for reactions that range from 20 °C to 25 °C (LEE; MEI;

DECKER, 1997; HERNÁNDEZ; PARK; RHEE, 2002). In the

present study, the rst stage comprised essays carried out at

22°C without pre-incubation. In the second stage, dierent

times and temperatures of pre-incubation were tested.

In the reaction catalyzed by the GSH-Px enzyme, the

oxidized glutathione (GSSG) generated from glutathione

(GSH) (Equation6) is instantly and continuously reduced in

the presence of the GSH-Px enzyme (Equation7). is keeps

a constant glutathione level, preventing the redox cycle from

stopping (ROVERJUNIORetal., 2001; PENHA-SILVAetal.,

2005).

22 2

2 –2

GSH H O GSH Px GSSG H O+→ → +

(6)

2GSSG NADPH H GS RED GSH NADP

+ +→ → +

(7)

In this redox cycle, the NADPH molecule behaves as an

electron donor, which is oxidized. In the standard Paglia and

Valentine (1967) method, this oxidation is photometrically

monitored by the absorption decay at 340nm, which is

correlated with the GSH-Px enzymatic activity. Figure2 shows

that the GSH-Px enzymatic activity during the ve minutes of

reaction is inuenced by the dierent pre-incubation conditions

of the reaction medium (22 °C immediate, 36 °C/30min,

and 36 °C/10min).The reaction rate was 0.0003 abs/min

when the sample was pre-incubated at 36°C for 30 minutes;

0.0007 abs/min for pre-incubation at 36°C for 10 minutes;

and 0.0058 abs/min for the reaction at 22°C without pre-

incubation. Such behavior could be attributed to the fact that

this enzymatic reaction is rather fast, probably taking place

Figure 1. Eect of dierent hydrogen peroxide concentrations on the

reaction rates (abs.min

–1

) of the conversion of hydrogen peroxide by

the GSH-Px enzyme for the immediate reaction at 22°C in raw chicken

thigh meat frozen for 120 days at –18 °C. e data are expressed as mean

± standard deviation (n = 3).

A,B,C

Dierent letters indicates signicant

dierence by the Tukey test (p < 0.05).

Figure 2. Inuence of dierent pre-incubation conditions of the

reaction medium on NADPH absorbance at 340nm (GSH-Px

enzymatic activity) during the reaction period. Symbols: ( )

immediate reaction at 22°C; () reaction aer water bath at 36°C for

30 minutes; (■) reaction aer water bath at 36°C for 10 minutes. e

data are expressed as mean±standard deviation (n = 3).

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

665

Cichoski et al.

4 Conclusions

e data obtained in this study help to elucidate the reaction

mechanisms that take place in the method used for measuring

the GSH-Px enzymatic activity. e dierent parameters of

enzymatic assay studied showed that the activity of GSH-Px

is inuenced by some parameters related to the analytical

method. When hydrogen peroxide was added at concentrations

above 0.72 mM, the GSH-Px enzyme was saturated and its

activity was inhibited. GSH-Px decreased its activity in contact

with terc-butil hydroperoxide when compared to hydrogen

peroxide, conrming its higher anity to the latter. Other

important ndings were that the highest GSH-Px activity was

observed when enzyme and substrate were put into contact at

22°C without any pre-incubation and that the use of reaction

media containing mercaptoethanol was not associated with an

increase the GSH-Px enzymatic activity, as previous reported

in literature. e results obtained allowed to dene the best

2007). In other words, GSH-Px enzyme presents higher anity

to hydrogen peroxide. e reaction rates of the conversion of

hydrogen peroxide and terc-butil hydroperoxide by GSH-Px

enzyme at 22 °C are shown in Figure3. e GSH-Px enzymatic

activity was 0.0028 abs/min for the conversion of terc-butil

hydroperoxide and 0.0077 abs/min for the conversion of

hydrogen peroxide conrming that GSH-Px has higher anity

to the latter. Avissaretal. (1991) found similar enzymatic activity

values of milk GSH-Px when using hydrogen peroxide and terc-

butil hydroperoxide as substrates.

Eect of the presence of dierent buers in the reaction medium

on the GSH-Px activity

e determination of GSH-Px enzyme activity in animal

tissues requires a previous extraction, since the enzyme is found

inside the mitochondria and cytosol of the muscle cells (CHAN;

DECKER, 1994). During extraction, some conditions must

be controlled in order to preserve the enzyme. ose factors

include pH and electrolytes concentration in the buer solution,

extraction temperature, and solution dilution (FENNEMA,

1993).

The GSH-Px optimal pH is close to 8.0 (PAGLIA;

VALENTINE, 1967), and its activity is minimal at pH lower

than 6.0 (MILLS, 1959). erefore, the GSH-Px activity is oen

measured at the 7.0-7.6 pH range (PUNCHARDetal., 1996).

High electrolyte concentrations aect the proteins solubility

(FENNEMA, 1993). According to Chen, Lindmark-Mansson

and Akesson (2000), low buer concentrations increase the

extraction of the GSH-Px enzyme. e extraction temperature

is also critical since high temperatures can cause thermal

inactivation of the enzyme. Thus, in this study, the buffer

solution was kept at 4°C, the homogenization took place under

ice bath, and the centrifugation was performed at 4°C.

The active center of GSH-Px contains the amino acid

selenocysteine, a derivative of cysteine in which a sulfur atom is

substituted by selenium. Selenocysteine facilitates the GSH-Px

oxidation by generating a covalent bond between two atoms

of selenium. ese bonds make the protein structure more

stableimpairing GSH-Px extraction and reducing its reactivity

with the substrate (LEHNINGER; NELSON; COX, 1995).

According to Araietal. (1994), mercaptoethanol is a stabilizing

agent which forms a reversible enzyme-inhibitor (FARFÁN,

1994), a useful characteristic when one wants to prevent

spontaneous oxidation during the GSH-Px enzyme extraction.

erefore, it was expected that the presence of mercaptoethanol

in the buer solution favored the enzymatic extraction. To study

the eect of dierent buer solutions on the GSH-Px enzymatic

activity, tris-HCl buer 50 mM pH 7.6 and two other types

of extraction buers containing mercaptoethanol were used.

e results are shown in Figure4. e GSH-Px enzymatic

activity was 0.0062 abs/min using an extraction medium

with buer number 1, 0.0072 abs/min using buer number 2,

and 0.0114abs/min using buer number 3. e results show

that the highest enzymatic activity was observed when buer

number 3 was used (tris-HCl 50 mM pH 7.6), suggesting that

mercaptoethanol was not useful for improving the enzyme

extraction. Chaudiereetal. (1984) observed inhibition of the

GSH-Px enzymatic activity when adding mercaptoethanol to the

reaction medium obtaining the same results hereby reported.

Figure 3. Eect of substrate type on NADPH absorbance at 340nm

(GSH-Px enzymatic activity) during the reaction period for the

immediate reaction at 22°C. Symbols: ( ) hydrogen peroxide

at 0.72mM; (■) terc-butil hydroperoxide at 15 mM. e data are

expressed as mean ± standard deviation.

Figure 4. Inuence of the presence of buers in the reaction medium

on NADPH absorbance at 340 nm (GSH-Px enzymatic activity)

during the reaction period for the immediate reaction at 22 °C.

Symbols: (■) buer 1 (potassium phosphate 50 mM at pH 7,0+EDTA

1mM+mercaptoethanol 1 mM); () buer 2 (tris-HCl 50 mM at pH

7,6+EDTA 1 mM+mercaptoethanol 5 mM); (♦) buer 3 (tris-HCl

50 mM pH 7,6). e data are expressed as mean ± standard deviation

(n = 3).

Ciênc. Tecnol. Aliment., Campinas, 32(4): 661-667, out.-dez. 2012

666

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