J Sci Food Agric 1996,70,75-78

Mechanism of Methylene Blue Bleaching by Lipox ygenase To s hi yuki To yo sa ki Department of Home Economics, Koran Women's Junior College, Yokote 1-2-1 Minami-ku, Fukuoka City, Fukuoka 816, Japan (Received 28 November 1994; revised version received 18 April 1995; accepted 2 August 1995)

Abstract: A lipid peroxidation reaction and methylene blue bleaching reaction are thought to proceed in the presence of methylene blue with linoleic acid as the substrate from the time lipoxygenase is added until methylene blue bleaching concludes. For 15 s after adding the lipoxygenase, neither the lipid peroxidation reaction nor the bleaching reaction occurred, but they both proceeded after that time period. The results suggested that hydroperoxide is first formed by the lipid peroxidation reaction, after which methylene blue specifically picks the hydrogen from the hydroperoxide 13 -00H isomers, and then the methylene blue is reduced.

Key words: methylene blue, bleaching, lipoxygenase, lipid peroxidation, hydro- peroxide isomers.

INTRODUCTION

Lipoxygenase (linoleate : oxygen oxydoreductase, EC 1.13.11.12) is widely distributed in plants and animals, and catalyzes the dioxygenation of the cis,cis-1,4-penta- diene structure in fatty acids (eg linoleic and linolenic acids) and fatty acid esters by molecular oxygen (Vliegenthart and Veldink 1982). This enzyme has nega- tive and positive effects on the quality of products from plant and animal origins. The lipoxygenase-mediated reaction contributes to the formation of off-flavour and decreases in nutritional value of food (Angel0 1992). Lipoxygenase activity is generally measured by moni- toring the enzyme reaction with linoleic acid as the sub- strate (Bonnet and Crouzet 1977; Cayrel et a1 1983; Daood and Biacs 1988; Olias and Valle 1988; Boyes et a1 1992; Piazza 1992).

Adding lipoxygenase in the presence of methylene blue with linoleic acid as the substrate, the methylene blue undergoes rapid bleaching after an initial delay. It has also been found that the interval between the addition of lipoxygenase and the bleaching of methylene blue is affected by lipoxygenase activity level (Toyosaki 1992a). In view of this fact, there is a negative corre- lation between the time at which the bleaching of methylene blue begins and lipoxygenase activation. Lipoxygenase activity can be calculated from the time

needed for the bleaching of methylene blue to begin (Toyosaki 1992a). The sequence of reactions thought to occur after the addition of lipoxygenase to the conclu- sion of methylene blue bleaching is a lipid peroxidation reaction and the reaction of methylene blue bleaching, but it is not clear how these two reaction mechanisms proceed. This report concerns a study of this sequence of events.

MATERIALS AND METHODS

Materials

Linoleic acid (more than 99.6% pure) was purchased from Nacalai Tesque, Inc (Kyoto, Japan). Lipoxygenase (from soya beans, Type I-B, lyophilised) was obtained from the Sigma Chemical Co (St Louis, MO, USA). Methylene blue (more than 95% pure) was obtained from Tokyo Kasei Co Ltd (Tokyo, Japan). Other reagents were of special grade and were obtained from Nacalai Tesque.

Preparation of reagents

The 0.2 M borate buffer was at pH 9.0. The substrate solution of 20 mM linoleic acid in borate buffer con- tained ethanol (3 ml litre-'). The methylene blue solu- tion was diluted with the borate buffer to 2.5 mM, and

75 J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain

76 T To yosaki

the lipoxygenase solution was diluted with the borate buffer to give solutions with activity of 500 units m1-l.

Reaction by lipoxygenase

The reaction was initiated by injecting 100 pl of working enzyme solution into a thermally equilibrated cuvette containing 1.0 ml of methylene blue solution and 1.0 ml of linoleic acid solution and 0.9 ml of borate buffer. The reaction mixture (total volume 3.0 ml) was stirred for 10 s after which absorbance at 660 nm was recorded by a spectorophotometer (Model V-2000, Hitachi Ltd, Tokyo, Japan) at intervals of 1 s for 60 s at 25°C.

Isolation of linoleic acid hydroperoxide

The reaction was stopped by adding sodium boro- hydride (10 mg) to stabilise the hydroperoxide. After acidification to pH 3.0, linoleic acid was extracted four times with an equal volume of diethyl ether, washed with distilled water until the washings became neutral, and dried over anhydrous sodium sulphate overnight.

Measurements of linoleic acid hydroperoxide

Linoleic acid hydroperoxide in the reaction mixture was monitored by measuring the increase in absorbance at 234 nm over a period of 20 min. The initial reaction velocity was calculated by linear regression using an Excel spreadsheet of the data over the interval between 20 and 60 s of the reaction curve (Toyosaki et al 1992b).

Isomeric analysis using HPLC

The amounts of hydroperoxide isomers in the reaction mixtures were determined according to the HPLC method of Mohri et a/ (1992), using a Radial-Pak silica cartridge 10 pm column (100 x 8 mm, Waters Associ- ates, Milford, MA, USA) at 40°C. The mobile phase was composed of n-hexane/2-propanol (90.3/0.7,v/v) at flow rate of 1.0 ml min-'. The eluant was monitored with a UV detector at 234 nm. The isomeric compositions of four peaks corresponding to 9-hydroperoxy-lO-E,12-Z- octadecadienoic acids (9-OOH(E,Z))/9-hydroperoxy-l0- E,12-Z-octadecadienoic acids (9-00H(E,E))/13-hydro- peroxy-9-EJ l-E-octadecadienic acids (13-00H(E,E))/ 13-hydroperoxy-9-Z,1 l-E-octadecadienoic acids (13- OOH(Z,E)) were established according to the method of Hamberg and Samuelsson (1967).

RESULTS AND DISCUSSION

When following the changes over time at 660 nm, at which methylene blue exhibits maximum absorption, a

\

0 1 , , , , , , . 0 5 10 15 20 25 30 35 40 45 50 55

Time (sec) Fig 1. Bleaching of methylene blue during lipid peroxidation by lipoxygenase. The reaction mixture contained 10 mM methylene blue and 20 mM linoleic acid in 0.2 M borate buffer. The reaction was started by adding lipoxygenase in a total volume 3.0 ml. One enzyme unit corresponds to the formation

of 1 pmol hydroperoxide min-'.

reaction sequence such as that shown in Fig 1 proceeds after adding lipoxygenase in the presence of methylene blue with linoleic acid as the substrate. Among this sequence of reactions is a lipid peroxidation reaction and a methylene blue bleaching reaction (Toyosaki 1992a). For this reason, the reaction sequence has been divided into overlapping intervals A (0-15 s), B (0-40 s), C (0-60 s), and note made of the types of reactions that proceeded in each. In intervals B and C, the reaction sequence was not stopped. Interval B subsumes A, and interval C subsumes A and B.

Interval A(15 s) began with the addition of lipoxyge- nase to the reaction solution. During this time interval, there is a negative correlation with the activation of lipoxygenase (Toyosaki 1992a). The kind of mechanism that proceeds at this stage was investigated. Because the methylene blue bleaching reaction does not occur during this interval, the lipid peroxidation reaction here was also investigated. One can confirm whether or not methylene blue is involved in the lipid peroxidation reaction by trying it with and without methylene blue. As Fig 2 shows, when methylene blue is present in the reaction solution, the lipid peroxidation reaction begins about 15 s after adding lipoxygenase. On the other hand, when methylene blue is not present the lipid per- oxidation reaction begins immediately after adding lipoxygenase; the amount of conjugated diene attains a maximum after about 15 s, and declines thereafter. These results suggest that during interval A the lipid peroxidation reaction does not occur in the presence of methylene blue, but does occur during intervals B and C.

Occurrence of the lipid peroxidation reaction during intervals B and C produces hydroperoxide. This hydro- peroxide is involved in the methylene blue bleaching reaction. This was shown by using HPLC, both when methylene blue was and was not present, during each of the intervals to determine the amount of hydroperoxide

Mechanism of methylene blue bleaching b y lipoxygenase 77

Fig 2. Changes of conjugated diene in the reaction mixtures with or without methylene blue during lipid peroxidation by

lipoxygenase. Each point represents the mean of four trials.

formed, and to ascertain from changes in this amount whether hydroperoxide is involved in the bleaching reaction. This research also included an investigation of how hydroperoxide is involved in the bleaching reaction at the hydroperoxide isomer level. Figure 3 shows an example of the result obtained with HPLC, which made it possible to separate out four hydroperoxide isomers. For the first 15 s after adding lipoxygenase, the hydro- peroxide peak could not be found when methylene blue was present, but the peak appeared when methylene blue was not present. Particularly evident was the peak for the 13 -00H (E,E) isomer. During an interval of 40 s after the addition of lipoxygenase, hydroperoxide was formed regardless of the presence or absence of methy- lene blue. Particularly evident was the peak for the 13-00H (Z ,E) isomer. Sixty seconds after the addition of lipoxygenase, peaks for 13 -00H (E,E and 2,E) appeared. Table 1 shows the results obtained when determining the amount of hydroperoxide isomer for each peak. During interval A (ie the first 15 s after adding lipoxygenase), hydroperoxide was not formed at

Time Lsec)

A (0- 15)

A +B (0-40)

With methylene blue Without rnethylene blue

I

, . .

o 10 20 30 40 0 10 20 30 40

Retention time (min)

Fig 3. Chromatograms of hydroperoxide isomers in the reac- tion mixtures with or without methylene blue during lipid per-

oxidation by lipoxygenase.

all in the presence of methylene blue, but it was formed when methylene blue was not present. Particularly, the amounts of 13 -00H isomers formed were very large. Hydroperoxide was formed during both interval B, lasting up to 40 s after adding lipoxygenase, and inter- val C (60 s), but although hydroperoxide was formed whether or not methylene blue was present, less was formed when methylene blue was present. This shows that in the presence of methylene blue the lipid peroxi- dation reaction does not occur for the first 15 s after adding lipoxygenase, thus strongly supporting the results shown in Fig. 1. Fifteen seconds after adding

TABLE 1 Changes of hydroperoxide isomer on the enzymic peroxidation of linoleic acid with or without methylene blue

Methylene blue Time (s) Hydroperoxide isomer (YO)

1 3 - 0 0 H (E,E) 1 3 - 0 0 H (Z,E) 9 - 0 0 H (E,Z) 9 - 0 0 H (E,E)

With A (0-15) 0" 0" 0" 0" A + B (0-40) 48.5 10.3 0.7 0 A + B + C (0-60) 92.1 4.6 1.2 0

Without A (0-15) 28.4 45.1 0.5 0 A + B (0-40) 93.5 23.7 0.7 0.2 A + B + C (0-60) 85.3 11.2 0.5 0.3

a Mean of three measurements, each made in duplicate.

78

90

80

70

60- h

C .s 50 2

4 0 -

" 30-

T Toyosaki

- -

-

-

loo r

"0 10 20 30 40 50

Methylene hlue concentration (mM) Fig 4. Effect of methylene blue concentration on the gener- ation of hydroperoxide isomers. Each point represents the

mean of four trials.

lipoxygenase, the lipid peroxidation reaction and methylene blue decolourising reaction begin, and because less hydroperoxide is formed when methylene blue is present than when it is not present, it was pos- sible to infer that the hydroperoxide formed is involved in the methylene blue bleaching reaction. In particular, it seemed that the hydroperoxide isomers 13 -00H were specifically involved. It is therefore possible to deter- mine if hydroperoxide, especially 13-00H isomers, is involved in the methylene blue bleaching reaction by investigating the changes in the amounts of hydro- peroxide isomers formed in relation to methylene blue concentration. Figure 4 shows that up to a methylene blue concentration of about 25 mM, the amounts of 13-00H isomers formed decreased almost linearly, but that they increased when the methylene blue concentra- tion exceeded 25 mM. By contrast, the amounts of 9 - 0 0 H isomers did not change at all in response to methylene blue concentration. This demonstrates that hydroperoxide (especially 13 -00H isomers) is specifi- cally involved in the methylene blue decolourising reac- tion.

There is a time interval during which no reactions occur at all after the addition of lipoxygenase, after which the lipid peroxidation reaction and bleaching reaction proceed. These two reactions apparently proceed according to the following mechanism.

lipoxygenase

LH -L* L . .LOO*

LOO. P LOOH (13-00H) + La

LOOH (13-00H) + MB 1 3 - 0 0 * + MB-H

This means that the lipid peroxidation reaction first occurs under the influence of lipoxygenase, forming hydroperoxide. Methylene blue specifically picks the hydrogen from the hydroperoxide 13 -00H isomers, and then the methylene blue (MB) is reduced, becoming MB-H. It would appear that this is the bleaching mechanism of MB.

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