studies on the inactivation of lysozyme by malonaldehyde

STUDIES ON THE INACTIVATION OF LYSOZYME BY MALONALDEHYDE

RAMESH CHANDER, S. V. SHEREKAR and M. S. GORE Biochemistry & Food Technology Division

Bhabha Atomic Research Centre Trombay, Bombay 400 085

India Received for Publication April 6, 1981

ABSTRACT

The interaction of malonaldehyde with lysozyme has been assessed with reference to incubation time, pH, concentration and aging. Gel filtration studies revealed that freshly prepared malonaldehyde contains a small proportion of compounds absorbing at 267 and 350 nm, which shows significant increase upon incubation of rnalonaldehyde. While higher concentration of mabnaldehyde leads to inactivation of enzyme, lower concentration (2.5 mM) stimulates the lytic function of lysozyme. Stimulation and inactivation o f the enzyme have been discussed in the context of degradation of malonaldehyde.

INTRODUCTION

Oxidation of unsaturated fatty acids leads to the formation of a number of thiobarbituric acid reacting compounds (Gutteridge et al. 1974), major one among them being malonaldehyde (Kwon and Olcott 196613). It is also produced during irradiation of amino acids such as arginine, glutamic acid, methionine, homoserine (Ambe and Tappel 1961) and other compounds like glycerol (Scherz 1968). Malonalde- hyde has been shown to interact with bovine plasma albumin (Craw- ford et al. 1967), myosin (Buttkus 1967) and DNA (Reiss et al. 1972). Ability of malonaldehyde to inhibit hydrolytic enzymes such as ribonuclease (Chio and Tappel 1969; Shin et al. 1972) and papain (Shin et al. 1972) has also been demonstrated. Besides, lipid peroxidation in vivo is known to cause deleterious effect on biological systems (Tappel 1973).

Malonaldehyde itself is not a stable compound (Kwon and Watts 1963). Different reactivities of malonaldehyde towards enzymes or biological systems may be expected particularly when the reaction

Journal of Food Biochemistry 5(1981) 323-324. All Rights Reserved @ Copyright 1982 by Food & Nutrition Press, Inc., Westport, Connecticut. 313

314 RAMESH CHANDER, S. V. SHEREKAR and M. S. GORE

occurs under conditions not favorable to the stability of malonaldehyde. This has not however been sufficiently examined. The present communication reports on the interaction of malonaldehyde with lysozyme in relation to concentration and the products formed.

MATERIALS AND METHODS

Lysozyme (Grade I, 3 x crystallized) and dried cells of Micrococcus Zysodeikticus were obtained from Sigma Chemical Co., U.S.A. 1,1,3,3- tetraethoxypropane was a product of Fluka A G, Switzerland. Sepha- dex G-10 was obtained from Pharmacia Fine Chemicals, Sweden. All other reagents were of analytical quality.

Preparation of Malonaldehyde Malonaldehyde was prepared from 1,1,3,3,-tetraethoxypropane

(TEP) by acid catalyzed hydrolysis of the acetal. The system con- tained 0.5 ml TEP, 0.5 ml of 0.1 N HCl and 9.0 ml of distilled water in a glass stoppered tube. It was held at 50°C for 45-50 min in a waterbath with intermittent shaking. After adding 2.5 ml of 0.1 N NaOH, the volume was made up to 50 ml with 0.05 M phosphate buffer, pH 6.2. Method described by Turner et at. (1954) was followed to estimate malonaldehyde. A suitably diluted aliquot (5 ml) was treated with 5 ml of 1 M orthophosphoric acid containing 0.36% thiobarbituric acid (TBA) and held in a boiling water bath for 45 min. Glass marbles were placed over the tubes to prevent evaporation losses. The tubes were cooled and the color was read at 535 nm on Bausch and Lomb Spectro- colorimeter. Standard graph was prepared using accurately weighed TEP.

Fractionation of Malonaldehyde Procedure described by Kwon and Olcott (1966a) was followed with

minor modifications. Sephadex G-10 column (1.3 cm x 70 cm) was equilibrated with 0.05 M phosphate buffer, pH 6.2 for 24 h prior to use. Void volume of the column was determined using blue dextran. One ml of malonaldehyde (10 mM) was applied and the column was eluted with phosphate buffer at a flow rate of 30 ml/h. Fractions (5.2 ml) were collected in LKB fraction collector, Ultrorac 7000 at 0-2°C. Absorbance of the fractions was recorded at 267 and 350 nm on Beckman DB spectrophotometer. Reactivity of the fractions with TBA was also measured as described earlier.

INACTIVATION OF LYSOZYME BY MALONALDEHYDE 315

Reaction of Malonaldehyde with Lysozyme

Alteration in the activity of lysozyme as induced by malonaldehyde with respect to its concentration, pH and incubation time and substrate concentration was determined. Incubation of lysozyme (0.5 mg/ml) and malonaldehyde in appropriate buffers was carried out in glass stoppered tubes kept in a n incubator at 37°C. Aliquots were withdrawn at desired intervals to estimate the activity of lysozyme.

Assay of Lysozyme

Lysozyme activity was assayed by measuring the decrease in optical density (0.D) of cell suspension of M. lysodeikticus. The initial O.D. of cell suspension in 0.05 M phosphate buffer, pH 6.2 was adjusted to 0.60 at 540 nm in Bausch and Lamb Spectrocolorimeter. The reaction mixture included 4 ml of cell suspension and 1 ml of lysozyme solution (2.5 pg/ml). Decrease in O.D. on incubation for 30 min at 37°C was recorded. On the basis of the known initial O.D. and final O.D. obtained at time ‘t’, percent decrease in O.D. was calculated.

RESULTS

Interaction of lysozyme with malonaldehyde over a period of 24 h at 37°C was examined in terms of changes in lysozyme activity as shown in Fig. 1. Activity of lysozyme incubated without malonaldehyde did not show appreciable decrease over a period of 24 h. It was retained to the extent of 93%. On the contrary, lysozyme incubated with 10 mM malonaldehyde showed a n increase in activity up to 10 h. Further incubation resulted in a progressive decline in the activity and only 27% of the original activity was retained after 24 h.

Effect of pH

Influence of pH on the alteration in the activity of lysozyme by malonaldehyde in the pH range of 4-8 using appropriate buffers (citrate-phosphate and sodium phosphate system) is depicted in Fig. 2. Maximum loss in the activity of lysozyme was observed between pH values of 5.5 to 6.2.

Effect of Concentration

Results pertaining to the effect of concentration of malonaldehyde on the lysozyme activity are presented in Fig. 3. As the concentration


125 c

FIG. 1. EFFECT OF MALONALDEHYDE ON LYSOZYME ACTIVITY AS A FUNCTION OF TIME

The enzyme (0.5 mg/ml) was incubated with 10 mM malonaldehyde at 37°C in 0.05 M phosphate buffer, pH 6.2. Lysozyme incubated similarly in absence of malonaldehyde served aa control. Aliquota were drawn at regular intervals and the activity was

determined. Activity is expressed as percent of control.

of malonaldehyde increased from 5-20 mM, decrease in the lysozyme activity was observed, the fall in activity being particularly steep between 5-10 mM. On the contrary, lysozyme incubated with low concentration of malonaldehyde (2.5 mM) led to rapid lysis of the substrate indicating stimulation of lysozyme activity. The stimula- tory effect was also observed when varying concentrations of the substrate, M. lysodeikticus cells, were used (Fig. 4). This effect was also observed at 1.25 mM concentration of malonaldehyde. Concen- tration lower than 1.25 mM did not influence the activity of lysozyme.

Degradation of Malonaldehyde

Since prolonged incubation period is needed to affect the activity of lysozyme by malonaldehyde, it becomes essential to investigate the


w 2o I > N 0

; i31 10

L 5 6 7 PH

FIG. 2. EFFECT OF pH ON MALONALDEHYDE-LYSOZYME INTERACTION The enzyme (0.5 mg/ml) was incubated at 37°C with malonaldehyde (10 mM) at various pH values in 0.05 M citrate-phosphate or phosphate buffer. Lysozyme incubated similarly in abence of malonaldehyde served as control. The enzyme activity was

determined after incubation for 24 h.

MALONALDEHYDE, mM

FIG. 3. EFFECT OF MALONALDEHYDE CONCENTRATION ON LYSOZYME ACTIVITY

Malonaldehyde at various concentrations was incubated at 37°C with lysozyme (0.5 mg/ml) in 0.05 M phosphate buffer, pH 6.2. The enzyme activity was determined after

incubation for 24 h.


80 s 0 0

f 70 w In a

W K u # 60-

-

-

1 I 1 0 0.1 0.2 0-3 0

50

SUBSTRATE. mg/ml b

FIG. 4. INFLUENCE OF SUBSTRATE CONCENTRATION ON LYSOZYME ACTIVITY:

Malonaldehyde at 2.5 mM concentration was incubated for 24 h at 37OC with lysozyme (0.5 mg/ml). Lysozyme incubated in absence of malonaldehyde served as control. Activity of the enzyme was determined over substrate concentrations ranging from 0.16 mg/ml to 0.32 mg/ml. Results are expressed in terms of percent decrease in O.D. caused

by lysozyme incubated with (0-0) or without (*-a) malonaldehyde.

stability of the reacting component under identical conditions. As shown in Fig. 5, considerable loss of malonaldehyde measured in terms of absorption at 267 nm occurred within 8 h of incubation. Further incubation did not lead to any appreciable decrease in O.D. at 267 nm. Decrease in absorbance was also accompanied by an increase in absorbance at 350 nm which was pH dependent. A marked increase in the absorption at 350 nm was observed when malonaldehyde was incubated at pH 4-5 for 24 h.

Influence of Aged Malonaldehyde on Lysozyme Since malonaldehyde undergoes degradation during the course of

incubation either alone or in presence of lysozyme, the efficacy of aged malonaldehyde in influencing the lysozyme activity was ascer- tained. As against the stimulation of enzyme activity by fresh malonaldehyde (10 mM) in 6 h, aged malonaldehyde caused decrease in activity as shown in Fig. 7. Enzyme inactivation to the extent of 50% was brought about by aged malonaldehyde within 9 h. For


0 ~ 4 0 10 20 21 48

TIME, hr

FIG. 5. CHANGE IN ABSORBANCE CHARACTERISTICS OF MALONALDEHYDE AFTER STORAGE

Malonaldehyde (20 mM) in 0.05 M phosphate buffer, pH 6.2 was incubated at 37OC. Aliquots were withdrawn at intervals and absorbance was measured at 267 nm and 350 nm after 2000 and 100 fold dilution, respectively. Similar trend was observed with a 10 mM solution but the extent of absorption at 350 nm is comparatively less.

similar inactivation of lysozyme by fresh malonaldehyde, an incubation period of 24 h was needed. Rapid inactivation of lysozyme was observed when lysozyme was treated with malonaldehyde at 20 mM concentration.

Fractionation of Malonaldehyde

Increased absorption of malonaldehyde at 350 nm, as a result of incubation at 37OC, suggested the formation of new products. Fresh or incubated malonaldehyde (37"C, 48 h) were, therefore, fractionated on Sephadex G-10 column. Elution profiles in terms of absorption at 267 nm, 350 nm and TBA reactivity of the fractions are shown in Fig. 6 a and b, respectively.

"he component eluting immediately after void volume (42 ml) showed reactivity with TBA but lacked absorption in UV or visible region. This component was probably unhydrolyzed TEP, since fresh and unhydrolyzed TEP showed identical elution profile. The second component showed strong absorption at 267 nm and reactivity with TBA but was devoid of absorption at 350 nm. This peak can be


FRACTION NUMBEff

FIG. 6a. ELUTION PATTERN OF MALONALDEHYDE ON SEPHADEX G-10 One ml of freshly prepared malonaldehyde (10 mM) or one ml from a 10 mM solution incubated at 37OC for 48 h was applied on sephadex G-10 column and the fractions (5.2 ml) collected. Absorbance of the fractions obtained from fresh (-) or incubated (--) malonaldehyde was read at 267 nm (0) and 350 nm (0) after appropriate dilutions, whenever necessary. Void volume of the column was 42 ml. Similar elution profile was

obtained when elution was carried out with buffer containing O.1M NaC1.

attributed to malonaldehyde. Malonaldehyde is characterized by a strong absorption at 267 and very weak absorption at 350 nm which is attributed to n-rr transition (Kwon and Van der Veen 1968). The latter peak is not detectable in a weak solution of malonaldehyde as is the case with eluted fractions. The third component showed weak absorp tion at 267 and 350 nm and proportionately lower TBA reactivity. Elution profile of aged malonaldehyde was qualitatively similar to that of fresh malonaldehyde but quantitatively different. The intens- ity of the first and second component in aged malonaldehyde showed appreciable decrease while third component showed a two-fold increase in absorption at 267 and 350 nm as well as TBA reactivity.

INACTIVATION OF LYSOZYME BY MALONALDEHYDE

26.0~

321

I

FRACTION NUMBER

FIG. 6b. TBA REACTIVITY OF FRACTIONS Reactivity of fractions from fresh (-)or incubated (-) malonaldehyde, as in Fig. 6, with TBA was examined using appropriately diluted aliquots. First peak in the figure corresponds to unhydrolyzed TEP and is devoid of absorption at 267 nm or 350 nm.

Thus, incubation of malonaldehyde resulted in the increased formation of products which absorbs at 267 and 350 nm.

Effect of Purified Malonaldehyde on Lysozyme

Considering the heterogenity associated with TEP hydrolysate, the purified malonaldehyde absorbing at 267 nm was obtained by fractionation on sephadex G10 column. This purified preparation (equiv- alent to 5 mM) failed to inactivate lysozyme over a 24 h incubation period. However, this fraction too was unstable and did result in products absorbing at 350 nm which then inactivated lysozyme. This suggests that products generated were also responsible for lysozyme inactivation.


I I 1 1 1

4 8 12 25 TIME, hr

FIG. 7. EFFECT OF AGED MALONALDEHYDE ON LYSOZYME ACTIVITY Freshly prepared malonaldehyde (--) and malonaldehyde aged for 24 h at 37OC (-) were incubated with lysozyme (0.5 mg/ml) at a concentration of 10 mM (-0) and 20 mM (-*).

DISCUSSION

Forgoing results indicate that lysozyme loses its activity as a consequence of interaction with malonaldehyde. However, inhibition of lysozyme activity by malonaldehyde requires fairly a long period of incubation. This assumes significance as malonaldehyde itself is reported to be unstable (Kwon and Watts 1963). The observed inhibition of lysozyme activity may also, therefore, be ascribed to the action of products resulting from malonaldehyde. This is apparent from the higher reactivity of aged malonaldehyde towards lysozyme. Despite loss in its absorption at 267 nm, aged malonaldehyde caused higher inhibition of lysozyme. These results are in agreement with the finding of Shin et al. (1972) on the inhibition of RNase activity by aged malonaldehyde.


Our results on the fractionation of malonaldehyde show that even the fresh preparation of malonaldehyde contains unhydrolyzed TEP and a small proportion of product with absorption at 267 and 350 nm. Recent studies of Marnett et al. (1979) using 14C labeled TEP cor- roborate our findings on the occurrence of three fractions in TEP hydrolysate. Presence of impurities in TEP hydrolysate may not permit to assess correctly the interaction of malonaldehyde with enzymes or other biological systems. Thus, studies of Marnett and Tuttle (1980) demonstrate that purified malonaldehyde is less muta- genic than P-hydroxyacridine, a product present in TEP hydrolystate.

Polymerization of protein by malonaldehyde might account for the loss of lysozyme activity as has been observed by Chi0 and Tappel (1969). It is worth noting that low concentrations of malonaldehyde activates lysozyme and that this contains comparatively low propor- tions of the products absorbing at 350 nm. Aiso, 10 mM of malonaldehyde accentuated lysozyme activity only in the early period of incubation when the proportion of the product was similarly low. This indicates that a low concentration of malonaldehyde or some product formed during incubation is likely to induce conformational changes in the structure of lysozyme favoring activation. Considering that malonaldehyde is a product of lipid peroxidation, it is relevant to note that the secondary products from linoleic acid hydroperoxides have been shown to activate pepsin activity (Gamage and Matsushita 1973).

In view of the higher reactivity of aged malonaldehyde towards lysozyme, it is essential to recognize the importance of degradation of malonaldehyde itself. Our studies using purified malonaldehyde obtained by sephadex G-10 chromatography show that malonaldehyde upon incubation becomes more potential in inactivating lysozyme, thus emphasizing the importance of degradation products.

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CHIO, K. S. and TAPPEL, A. L. 1969. Inactivation of ribonuclease and other enzymes by peroxidising lipids and by malonaldehyde. Biochemistry 8, 2827-2832.

CRAWFORD, D. L., YU, T. C. and SINHUBER, R. 0. 1967. Reaction of malonaldehyde with protein. J. Food Sci. 32, 332-335.

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GAMAGE, P. T. and MATSUSHITA, S. 1973. Interaction of autoxidised products of linoleic acid with enzyme proteins. Agr. Biol. Chem. 37, 1-8.

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KWON, T. W. and WA?T,S B. M. 1963. Determination of malonaldehyde by ultraviolet spectrophotometry. J. Food Sci. 28, 627-630.

KWON, T. W. and OLCOTT, H. S. 1966a. Malonaldehyde from the autoxida- tion of methyl linoleate. Nature 210, 214-215.

KWON, T. W. and OLCOTT, H. S. 1966b. Thiobarbituric-acid-reactive substances from autooxidised or ultraviolet-irradiated unsaturated fatty esters and squalene. J. Food Sci. 31, 552-557.

KWON, T. W. and VAN DER VEEN, J. 1968, Ultraviolet and infrared absorption spectra of malonaldehyde in organic solvents. J. Agr. Food Chem. 16,

MARNETT, L. J., BIENKOWSKI, M. J., RABAN, M. and TUTTLE, M. A. 1979. Studies on the hydrolysis of '%-labeled tetraethoxypropane to malonaldehyde. Anal. Biochem. 99, 458-463.

MARNETT, L. J. and TUTTLE, M. A. 1980. Comparison of the muta- genicities of malondialdehyde and the side products formed during its chemical synthesis. Cancer Res. 40, 276-282.

REISS, U., TAPPEL, A. L. and CHIO, K. S. 1972. DNA malonaldehyde reaction: formation of fluorescent products. Biochem. Biophys. Res. Commun. 48, 921-926.

SCHERZ, H. 1968. Uber die bildung von malondialdehyd bei du bestrahlung von glycerin. Experientia 24, 420-421.

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TURNER, E. W., PAYNTER, W. D., MONTIE, E. J., BESSERT, M. W., STRUCK, G. M. and OLSON, F. C. 1954. Use of 24hiobarbituric acid reagent to measure rancidity in frozen pork. Food Technol. 8, 326-330.

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studies on the inactivation of lysozyme by malonaldehyde

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