Toxicon, 1976, Vol.14,pp.35-42. Pergamon Press. Printed inGreatBritain.
TEMPERATURE
STABILITY OF PHOSPHOLIPASE A ACTIVITY-I
BEE (APIS MELLIFERA) VENOM PHOSPHOLIPASE A, CHITRA NAIR, JAN HERMANS, D. MUNJAL* and W. B. ELLIOTT Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14207, U.S.A. and Department
of Biochemistry, University of North Carolina, Chapel Hill, N.C. 27514, U.S.A. (Accepted
for publication
28 July 1975)
CHITRA NAIR, JAN HERMANS, D. MUNJALand W. B. ELLIOTT.Temperature stability of phospholipase A activity-I. Bee (Apis mellifera) venom phospholipase Al. Toxicon 14, 35-
1976.-Enzymic activity of bee venom phospholipase A, has been studied at high temperatures using dispersed egg yolk and sonicated dipalmitoyl and dicaproyl lecithins as substrates. With egg yolk substrate the maximum activity was observed at 65°C and both dipalmitoyl and dicaproyl lecithins gave a maximum at 60°C. At 60°C the enzyme showed loss of activity in the presence of 0.7 M urea. The loss of activity at cu. 60°C could be due to the physical change in the nature of the substrates and/or due to the denaturation of enzyme at this temperature. Preincubationof enzyme at temperatures up to 90°C for 45 min gave the same temperature optimum of 65°C with egg yolk substrate which shows that the heat denaturation, if it occurs, is reversible. O.R.D. studies of pure bee venom phospholipase Az showed no change in optical rotation from 10 to 9O’C. In spite of the constant optical rotatory dispersion of the protein up to 9O”C, sudden loss of enzyme activity was noticed above 60°C with the lecithin substrates.
42,
INTRODUCTION
PHOSPHOLIPASEA, (PLA,) (E.C. 3.1.1.4) hydrolyses its natural substrates into the corresponding lyso compounds with the release of free fatty acid. The enzyme activity has been investigated by several authors using different techniques and substrates (FAIRBAIRN, 1945; HANAHAN, 1952; HANAHAN et al., 1954; ROHOLT and SCHLAMOWITZ, 1961; ZAMECNIK et al., 1947; HABERMANN, 1957; MAENO et al., 1962; CONDREA et al., 1964; HUGHES, 1935 LBNG and PENNY, 1957; MAGEE and THOMPSON, 1960; BROWN and BOWLES, 1966; AUGUSTYN and ELLIOTT, 1969). Recently, DE HAAS et al. (1968) have determined the activity by acidimetric titration using the method of DESNUELLE et al. (1955) with egg yolk as the substrate. The variety of assay systems, nature of the substrates and assay mediums have yielded conflicting results and made interpretation of data difficult. Wu and TINKER (1969) and MUNJAL and ELLIOTT (1972) have reported the optimum temperature of PLA, activity to be ca. 50°C with dipalmitoyl lecithin as substrate. High conformational stability of the enzyme was also shown by SCANU et al. (1969) who have *Current address: Kentucky, U.S.A.
Department
of Pathology,
School of Medicine, 3.5
TOXICON
1976
Vol. 14
University
of Kentucky,
Lexington,
36
CHITRA NAIR, JAN HERMANS,
D. MUNJAL and W. B. ELLIOTT
shown that porcine pancreatic PLA, has cu. 50% helical content which did not change in 8 M urea. Although many studies have been made on PLA, from different sources and its resistance to denaturation on boiling, scanty information is available regarding the enzymic activity at high temperatures. The present investigation, therefore, was undertaken to establish that the enzyme may not be biologically active at elevated temperatures even though some of its physical properties remain unchanged at these temperatures. MATERIALS
AND METHODS
Activity of crude bee venom, or pure bee venom PLA,, was determined by automatic titration of the released fatty acid with 0.005 M NaOH in a thermostated micro reaction vessel (Radiometer, Copenhagen; pH stat model TTll/SBR12/ABUl2/T’TA31) at temperature up to 80°C (at pH 8.0, unless otherwise stated) using the method of DE HAAS etal.(1968) with the addition of 0.5 % (final concentration) bovine albumin to the reaction mixture. The action of PLAz on three types of substrates (egg yolk, dipalmitoyl lecithin, dicaproyl lecithin) was investigated. Aqueous emulsions of egg yolk were prepared each day by homogenizing one egg yolk in 200 ml of water using a Waring blender (high speed) for 60 sec. The reaction medium contained 5.0 ml of egg yolk suspension per 10 ml of assay medium with the final concentration of sodium deoxycholate, CaCl, and bovine serum albumin (Pentex, fatty acid poor) being 1.3 x 10ms M, 3 x 10e8 M and 0.5 %, respectively. Dipalmitoyl lecithin, 50 mg (General Biochemicals) was dissolved in 2 : 1 chloroform-methanol solution, flash evaporated to dryness and diluted to 10 ml in the assay medium with the final concentrations of sodium deoxycholate, CaCl, and bovine serum albumin being 1.3 x lOma M, 3 x lows M and 0.5x, respectively. The above solution was sonicated in order to get a better dispersion. Sonification (120 min) was under N, atmosphere and at low power in order to prevent oxidative degradation of lecithin. Sonicated aqueous dispersion of lecithin was checked for the presence of free fatty acid by a thin layer chromatography method (DITTMERand LISTER,1964). Dicaproyl lecithin (Serdary Research Lab., London, Ont., Canada) contained 40 mg of lecithin per ml of chloroform. The assay medium contained 0.5 ml (20 mg) dicaproyl lecithin evaporated to dryness and dissolved in 10 ml of assay medium (1.3 x lows M sodium deoxycholate, 3 x 10ea M CaCl, and O-5% bovine serum albumin) for the assay. The specific activity was calculated as the umoles of alkali consumed (equivalent to micromoles fatty acid released) per mg of protein or venom (dry weight) per minute. Pure PLA, was isolated from bee venom by the methods of MUNJAL and ELLIOTT(1971) and SHEPHERDetnl.(1974). All chemicals used were analytical grade. RESULTS
O.R.D.
studies
The optical rotatory dispersion curve of pure bee venom PLA,, as shown in Fig. 1, indicated both by its shape and by the value of specific rotation at 234 nm (a = -5200) that this enzyme possesses some a-helical structure, albeit not very much. The curve shown here is very similar to the one determined for snake venom PLAz by KAWAUCHI et al. (1971) both enzymes having a less extreme negative rotation near 234 nm than the porcine enzyme studied by SCANU et al. (1969). Hence the observation of constant optical rotation at 234 nm at temperatures from 10 to 90°C with a 1 cm thermostated cell containing a solution of pure PLA,, is good evidence that no gross conformational change takes place up to that temperature. No change in specific rotation occurred on changing the solvent from water to 50% alcohol. Activity
measurements
The reaction showed zero order kinetics and essentially a linear relationship was observed between the velocity of the substrate breakdown and the amount of enzyme used during the time necessary for titration equivalent to full scale deflection on the titrimeter chart as long as the quantity of fatty acid released per unit time does not exceed the maximum titration rate of the titrimeter. Uniform and vigorous stirring was necessary to prevent variable results. TOXICON
1976 Vol. II
Temperature
37
Stability of Phospholipase-I
x,
nm
FIG. 1. OPTICAL ROTATORY DISPERSION OF ISOLATED BEE VENOM PHOSPHOLIPASE TEMPERATURE, MEASURED WITH A CARY 60 RECORDING SPECIROPOLARIMETER, STANDARD CELL (0496mg PROTEINPER ml).
Aa
AT ROOM USING 1 CITl
Figure 2 shows the activity of crude bee venom PLA, and pure PLA, at high temperatures when egg yolk was used as the substrate. A linear rise in enzymic activity occurred with the increase in temperature and at 65°C the activity was maximal. Further increase in temperature resulted in a sudden decrease in activity to cu. 4% of the maximum activity, at 75°C. The same temperature optimum was noted when the egg yolk dispersion, which was heated to 90°C for 10 min, cooled and reblended, was used as the substrate or when hard
30
40
60
50
70
80
“C
FIG.
2.
EFFECT OF TEMPERATURE
(-+-•--);
TOXICON
1976 Vol. 14
ON THE ACTIVITY OF CRUDE ACCIVITY.
and purified bee venom PLA, activity -O-O--. dispersion.
BEE VENOM
PHOSPHOLIPASE
Substrate
AB
was egg yolk
CHITRA
38
NAIR,
JAN HERMANS,
D. MUNJAL
1
1000
40
50
and W. B. ELLIOTT
I
I
60
70
"C
FIG. 3. EFFECT OF TEMPERATURE ON PIIRIFIEDBEE VENOM PHOSPHOLIPASE A, ACTIVITY USING SONICATEDDIPALMITOYL LECITHIN,(--0--0-) AND DICAPROYL LECITHIN(-.-.--).
boiled egg yolks were dispersed instead of raw egg yolk. However, at 80°C the enzyme showed about 2% of the maximum activity with the addition of a highly concentrated solution of PLA,. The activity of pure bee venom PLA, with both sonicated dipalmitoyl lecithin and dicaproyl lecithin showed a maximum activity at 60°C in both the cases (Fig. 3). The activity of PLA, (egg yolk substrate) was totally suppressed by the addition of 10 ul of specific antiserum (rabbit) to the enzyme (prepared by immunization with pure enzyme) and incubating the mixture for 15 min at 37°C. Serum, up to 100 pl, from an
ii,
, 64
68
/
,
72
76
, 8.0
PH FIG. 4. EFFECTOF~H
TOXICON
1976 Vol. 14
AT~~~CONCRUDEBEEVENOMPHOSPHOLIPASEA~ACTIVITY. Substratewas egg yolk dispersion.
Temperature
Stability of Phospholipase-I
39
unimmunized rabbit did not inhibit the activity. This observation indicates that the measured activity was due to PLA, only. As shown in Fig. 4, the pH profile of the reaction at 65°C showed a maximum activity at pH 7-O as contrasted to a pH optimum of 8-O at room temperature. The effect of urea on the activity of PLA, at higher temperatures using egg yolk as substrate is shown in Fig. 5. At 40 and 50°C there is no change in the activity of PLA, with the addition of up to 2 M urea but with a higher concentration of urea a sudden loss of activity was noticed and the activity was completely lost in the presence of 4 M urea. At 60°C the activity loss was observed on addition of O-7 M urea and the enzyme became completely inactive with 2 M urea. Table 1 shows the effect of ethanol on the activity of phospholipase A, at 30 and 40°C. At 3O”C, an appreciable increase in activity was observed with lo,20 and 30% ethanol concentration but the activity became 0 at 40% ethanol. The activity was almost doubled with 10 and 20% ethanol at 40°C but was reduced with 30%
FIG. 5. EFFECTOF UREAON PUREBEEVENOMPHOSPHOLIPASE A, ACTIVITY. 0 4O”C, A 5O”C, !J 60°C. Substrate was egg yolk dispersion.
TABLE 1. EFFECTOF ETHANOLON THE PHOSPHOLIPASE A, ACTMTY
Alcohol concentration (%) 0 10 20 30 40
pmole of fatty acid released/mg of proteinjmin 30°C 40°C 312 550 444 1190 444 1190 432 680 0 0
Substrate used was egg yolk dispersion. TOXICON
1976 Vol. 14
40
CHITRA NAIR, JAN HERMANS,D. MUNJAL and W. B. ELLIOTT
ethanol and became zero at 40 ‘A. Due to the evaporation of alcohol at higher temperatures, the effect of alcoho1 on PLA, activity couId not be studied above 40°C. DISCUSSION Previous studies employing the hydroxamic acid assay with dipalmitoyl lecithin, dispersed by vortex mixing, as substrate showed the optimum temperature for bee venom PLA, activity to be 50°C (MUNJALand ELLIOTT,1972) which is in close agreement withthe work of WV and TINKER(1969) with PLA, from Crotdus atrox venom. When egg yolk was used as a substrate, PLA, showed an abrupt loss of activity above 65°C. Similarly, sudden loss of activity was observed with sonically dispersed dipalmitoyl lecithin, and dicaproyl lecithin at 60°C. This loss of activity could possibly be due to the abrupt change in the physical nature of the substrate, as the flocculation of the substrates starts at these temperatures. The above observations suggest that one of the reasons for the loss of activity could be the nonavailability of the substrate to enzyme due to a change in physical state of the substrate. However, the study on the effect of urea on the enzymic activity at elevated temperatures shows no loss of activity at 40 and 50°C in the presence of urea concentration up to 2 M, whereas at 60°C the enzyme starts losing its activity with the addition of urea to O-7 M and the activity becomes zero, at a urea concentration of 2 M. The increased sensitivity of the enzyme to urea at 60°C could indicate a weakening of a possible hydrophobic bonding critical to maintenance of the conformation of the active site. This suggests that the loss of activity at about 65°C might be due not only to the possible change in the physical nature of the substrate but could be due to a minor change(s) in conformation of the enzyme localized at or near the active site and/or the substrate recognition site. If such a change occurs on heating, it is reversible as the enzyme preheated without substrate at 90°C for 15 min at pH 8.0 folIowed by cooling also gave an optimum temperature of 65°C with egg yolk substrate. However, in the presence of cu. 1 M urea the incubation of the enzyme at 90°C for 15 min resulted in a total loss of activity. Earlier studies of heat stability of the enzyme were usually performed at pH 5.9 (HUGHES, 1935). However, the O.R.D. spectra of pure bee venom PLAz remained unchanged even at 90°C. In the presence of ethano1 at 30 and 4O”C, there was considerable change in the activity of PLA,. With 10 and 20% ethanol concentration increased activity was observed at 30 and 40°C and with 30% ethanol the reduction of activity was noticed in both the temperatures. But the decrease in activity with 30 % ethanol was greater at 40°C (ca. 45 % of the activity in 20% ethanol) than at 30°C (2%). This marked decrease in activity with a 10°C difference in temperature is quite significant, even though our results from O.R.D. studies showed no change in specific rotation in 50% ethanol at room temperature. Since the O.R.D. spectra show much less helical content for bee venom phospholipase A, than is present in porcine pancreatic phospholipase A,, it is not unexpected that the change in the biological properties of the enzyme due to reversible heat denaturation apparently did not cause any significant change in the optical rotatory dispersion of the - enzyme protein (measured in the absence of substrate). At 80°C the enzyme showed about 2 % of the maximum specific activity when measured with a highly concentrated solution of PLA, and egg yolk substrate. The catalytic activity of the enzyme is low at this temperature, and cannot be detectable with the usual low concentrations of the enzyme used in the assay at lower temperatures. TOXICON
1976 Vol. 14
Temperature
Stability of Phospholipase-I
41
The possible causes for loss of PLA, activity at elevated temperatures are : (1) a change in the substrate that makes it unavailable to the catalytic site of the enzyme and/or to the micelle recognition site of the enzymes which is necessary for maximal activity, (2) a minor localized reversible change in the conformation of the enzyme molecule which changes the catalytic activity by altering the active site but does not change the O.R.D. spectrum, (3) a loss of the micelle recognition site because of a reversible limited local conformational change, (4) an action of some other agent in the venom, on either the substrate or the PLA,, which has a very steep activation curve at high temperatures. In the case of egg yolk substrate, conceivably denaturation of the lipoprotein would render it unsuitable as a substrate; however, egg yolk substrate prepared from hard boiled egg yolks gave the same temperature optimum as did egg yolk substrate heated to 9O“C, allowed to flocculate and redispersed. The action of other factors in the venom on either the substrate or PLA, was eliminated by using pure PLA,. Since both the substrate dispersion structure and the conformation of the enzyme are stabilized by hydrophobic bonding, the experiments with urea and ethanol do not yield evidence clearly in support of any of the possibilities. However, heating the enzyme to 90°C in the absence of substrate or urea did not result in a loss of activity measured at lower temperatures, while heating to 90°C in the presence of 1 M urea resulted in a loss in activity when measured at lower temperatures, clearly indicating an action of urea on the enzyme molecule at high temperatures. At room temperatures ethanol up to 50% (v/v) produced no alteration in O.R.D. spectra; however, ethanol stimulated the hydrolysis of phospholipid. At WC, ethanol (10 and 20% v/v) was stimulating but 40% was totally inhibiting. This sharp loss in activity would seem to favor a change in the enzyme molecule as other factors affecting disperions, e.g. sonication, do not cause a total loss of activity at this temperature. Additional evidence favoring conformational change in the enzyme as the basis for the loss in activity is presented in the following paper (NAIR etal., 1976). Acknowledgement-The authors thank Mr. G. W. SHEPHERD for the purification of bee venom PLAl and Dr. JOHN WYPCH for a generous gift of specific antisera. This work was supported by a U.S.P.H.S. grant GM06241 and a fellowship to C.N. from the United Health Foundation. REFERENCES AUGUST~N,J. M. and ELLKIIT, W. B. (1969) A modified hydroxamate
assay of phospholipase A activity. Anal. Biochem. 31,246. BROWN,J. H. and BOWLES,M. E. (1966) Studies on the phospholipase A activity of Crotalus atrox venom. Toxicon 3,205. CONLIREA, E., DEVRIES,A. and MAGER,J. (1964) Hemolysis and splitting of human erythrocytes phospholipids by snake venoms. Biochim. biophys. Acta 84,60. DEHAAS, G. H., POSTEMA, N. M., NIEUWENHUIZ EN, W. and VAN DEENEN,L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas. Biochim. biophys. Acta 159,103. DESNIJELLE, P., CONSTANTIN, M. H. and BALDY, J. (1955) Technique potentiometrique pour la mesure de l’activitk de la lipas pancreatique. BUN. Sot. Chim. biol. 37,285. DITTMER,J. C. and LISTER,R. L. (1964) A simple specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid Res. 5,126. FAIRBAIRN,D. J. (1945) The phospholipase of the venom of the cotton-mouth moccasin (Agkistrodon piscivorous L.). J. biol. Chem. 157, 633. HABERMANN, E. (1957) Pharmacology of phospholipase A. Archs exp. Path. Pharmac. 230,538. HANAHAN,D. J. (1952) The enzymatic degradation of phospharidyl choline in diethyl ether. J. biol. Chem. 195, 199. HANAHAN,D. J., RODBELL,M. and TURNER,L. D. (1954) Enzymatic formation of monopalmitoleyl- and monopalmitoyl lecithin (lyso lecithins). J. biol. Chem. 206,431. HUGHES,A. (1935) The action of snake venoms on surface films. Biochem. J. 29,437. TOXICON
1976 Vol. 14
42
CHITRA NAIR, JAN HERMANS, D. MUNJAL and W. B. ELLIOTT
KAWAUCHI,S., IWANAGA,S., SAMEI~MA,Y. and Suzurcr, T. (1971) Isolation and characterization of two phospholipase A’s from the venom of Agkistrodon halys blornhofi. Biochim. biophys. Acta 236,142. LONG, C. and PENNY, I. F. (1957) The structure of naturally occurring nhosuhonlycerides. 3. Action of mocassin-venom phospholipase A on ovolecithin and related substat.& BioEhem.J. 65, 382. MAENO,H., M~~~IJHASKI,S., OKONOGI,T., HOSHI, S. and HOMMA,M. (1962) Studies on habu snake venom. V. Myolysis caused by phospholipase A in habu snake venom. Jap. J. exp. Med. 32,55. MAGEE,W. L. and THOMPSON,R. H. S. (1960) The estimation of phospholipase A activity in aqueous systems. Biochem. J. 77, 526. MUNJAL,D. and ELLIOTT,W. B. (1971) A simple method for the isolation of a phospholipase A from honey bee (Apis mellifera) venom. Toxicon 9,403. MUNIAL, D. and ELLIOTT,W. B. (1972) Further studies on the properties of phospholipase A from honey bee (Apis mellifera) venom. Toxicon 10, 367. NAIR, B. C., NAIR, C. and ELLIO~~, W. B. (1976) Temperature stability of phospholipase A activity-II. Variations in optimum temperature of phospholipases Aa from various snake venoms. Toxicon 14, 43. ROHOLT,0. A. and SCHLAMOWITZ, M. (1961) Studies of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A. Archs Biochem. Biophys. 94,364. SCANU,A. M., VAN DEENEN,L. L. M. and DE HAAS, G. H. (1969) Optical rotatory dispersion and circular dicroism of PLA, and its zymogen from porcine pancreas. Biochim. biophys. Acta 181,471. SHEPHERD,G. W., E~~orr, W. B. and ARBESMAN,C. E. (1974) Fractionation of bee venom. I. Preparation and characterization of four antigenic components. Prep. Biochem. 4,71. Wu, T. W. and TINKER,D. 0. (1969) Phospholipase Aa from Crotalus atrox venom. I. Purification and some properties. Biochemistry 8, 1558. ZAMECNIK,P. C., BREWSTER,L. E. and LIPMAN,F. A. (1947) Manometric method for measuring the activity of the Cl. welchi lecithinase and a description of certain properties of the enzyme. J. exp. Med. 85,381.
TOXICON
I976 Vol. I4
TEMPERATURE
STABILITY OF PHOSPHOLIPASE A ACTIVITY-I
BEE (APIS MELLIFERA) VENOM PHOSPHOLIPASE A, CHITRA NAIR, JAN HERMANS, D. MUNJAL* and W. B. ELLIOTT Department of Biochemistry, State University of New York at Buffalo, Buffalo, New York 14207, U.S.A. and Department
of Biochemistry, University of North Carolina, Chapel Hill, N.C. 27514, U.S.A. (Accepted
for publication
28 July 1975)
CHITRA NAIR, JAN HERMANS, D. MUNJALand W. B. ELLIOTT.Temperature stability of phospholipase A activity-I. Bee (Apis mellifera) venom phospholipase Al. Toxicon 14, 35-
1976.-Enzymic activity of bee venom phospholipase A, has been studied at high temperatures using dispersed egg yolk and sonicated dipalmitoyl and dicaproyl lecithins as substrates. With egg yolk substrate the maximum activity was observed at 65°C and both dipalmitoyl and dicaproyl lecithins gave a maximum at 60°C. At 60°C the enzyme showed loss of activity in the presence of 0.7 M urea. The loss of activity at cu. 60°C could be due to the physical change in the nature of the substrates and/or due to the denaturation of enzyme at this temperature. Preincubationof enzyme at temperatures up to 90°C for 45 min gave the same temperature optimum of 65°C with egg yolk substrate which shows that the heat denaturation, if it occurs, is reversible. O.R.D. studies of pure bee venom phospholipase Az showed no change in optical rotation from 10 to 9O’C. In spite of the constant optical rotatory dispersion of the protein up to 9O”C, sudden loss of enzyme activity was noticed above 60°C with the lecithin substrates.
42,
INTRODUCTION
PHOSPHOLIPASEA, (PLA,) (E.C. 3.1.1.4) hydrolyses its natural substrates into the corresponding lyso compounds with the release of free fatty acid. The enzyme activity has been investigated by several authors using different techniques and substrates (FAIRBAIRN, 1945; HANAHAN, 1952; HANAHAN et al., 1954; ROHOLT and SCHLAMOWITZ, 1961; ZAMECNIK et al., 1947; HABERMANN, 1957; MAENO et al., 1962; CONDREA et al., 1964; HUGHES, 1935 LBNG and PENNY, 1957; MAGEE and THOMPSON, 1960; BROWN and BOWLES, 1966; AUGUSTYN and ELLIOTT, 1969). Recently, DE HAAS et al. (1968) have determined the activity by acidimetric titration using the method of DESNUELLE et al. (1955) with egg yolk as the substrate. The variety of assay systems, nature of the substrates and assay mediums have yielded conflicting results and made interpretation of data difficult. Wu and TINKER (1969) and MUNJAL and ELLIOTT (1972) have reported the optimum temperature of PLA, activity to be ca. 50°C with dipalmitoyl lecithin as substrate. High conformational stability of the enzyme was also shown by SCANU et al. (1969) who have *Current address: Kentucky, U.S.A.
Department
of Pathology,
School of Medicine, 3.5
TOXICON
1976
Vol. 14
University
of Kentucky,
Lexington,
36
CHITRA NAIR, JAN HERMANS,
D. MUNJAL and W. B. ELLIOTT
shown that porcine pancreatic PLA, has cu. 50% helical content which did not change in 8 M urea. Although many studies have been made on PLA, from different sources and its resistance to denaturation on boiling, scanty information is available regarding the enzymic activity at high temperatures. The present investigation, therefore, was undertaken to establish that the enzyme may not be biologically active at elevated temperatures even though some of its physical properties remain unchanged at these temperatures. MATERIALS
AND METHODS
Activity of crude bee venom, or pure bee venom PLA,, was determined by automatic titration of the released fatty acid with 0.005 M NaOH in a thermostated micro reaction vessel (Radiometer, Copenhagen; pH stat model TTll/SBR12/ABUl2/T’TA31) at temperature up to 80°C (at pH 8.0, unless otherwise stated) using the method of DE HAAS etal.(1968) with the addition of 0.5 % (final concentration) bovine albumin to the reaction mixture. The action of PLAz on three types of substrates (egg yolk, dipalmitoyl lecithin, dicaproyl lecithin) was investigated. Aqueous emulsions of egg yolk were prepared each day by homogenizing one egg yolk in 200 ml of water using a Waring blender (high speed) for 60 sec. The reaction medium contained 5.0 ml of egg yolk suspension per 10 ml of assay medium with the final concentration of sodium deoxycholate, CaCl, and bovine serum albumin (Pentex, fatty acid poor) being 1.3 x 10ms M, 3 x 10e8 M and 0.5 %, respectively. Dipalmitoyl lecithin, 50 mg (General Biochemicals) was dissolved in 2 : 1 chloroform-methanol solution, flash evaporated to dryness and diluted to 10 ml in the assay medium with the final concentrations of sodium deoxycholate, CaCl, and bovine serum albumin being 1.3 x lOma M, 3 x lows M and 0.5x, respectively. The above solution was sonicated in order to get a better dispersion. Sonification (120 min) was under N, atmosphere and at low power in order to prevent oxidative degradation of lecithin. Sonicated aqueous dispersion of lecithin was checked for the presence of free fatty acid by a thin layer chromatography method (DITTMERand LISTER,1964). Dicaproyl lecithin (Serdary Research Lab., London, Ont., Canada) contained 40 mg of lecithin per ml of chloroform. The assay medium contained 0.5 ml (20 mg) dicaproyl lecithin evaporated to dryness and dissolved in 10 ml of assay medium (1.3 x lows M sodium deoxycholate, 3 x 10ea M CaCl, and O-5% bovine serum albumin) for the assay. The specific activity was calculated as the umoles of alkali consumed (equivalent to micromoles fatty acid released) per mg of protein or venom (dry weight) per minute. Pure PLA, was isolated from bee venom by the methods of MUNJAL and ELLIOTT(1971) and SHEPHERDetnl.(1974). All chemicals used were analytical grade. RESULTS
O.R.D.
studies
The optical rotatory dispersion curve of pure bee venom PLA,, as shown in Fig. 1, indicated both by its shape and by the value of specific rotation at 234 nm (a = -5200) that this enzyme possesses some a-helical structure, albeit not very much. The curve shown here is very similar to the one determined for snake venom PLAz by KAWAUCHI et al. (1971) both enzymes having a less extreme negative rotation near 234 nm than the porcine enzyme studied by SCANU et al. (1969). Hence the observation of constant optical rotation at 234 nm at temperatures from 10 to 90°C with a 1 cm thermostated cell containing a solution of pure PLA,, is good evidence that no gross conformational change takes place up to that temperature. No change in specific rotation occurred on changing the solvent from water to 50% alcohol. Activity
measurements
The reaction showed zero order kinetics and essentially a linear relationship was observed between the velocity of the substrate breakdown and the amount of enzyme used during the time necessary for titration equivalent to full scale deflection on the titrimeter chart as long as the quantity of fatty acid released per unit time does not exceed the maximum titration rate of the titrimeter. Uniform and vigorous stirring was necessary to prevent variable results. TOXICON
1976 Vol. II
Temperature
37
Stability of Phospholipase-I
x,
nm
FIG. 1. OPTICAL ROTATORY DISPERSION OF ISOLATED BEE VENOM PHOSPHOLIPASE TEMPERATURE, MEASURED WITH A CARY 60 RECORDING SPECIROPOLARIMETER, STANDARD CELL (0496mg PROTEINPER ml).
Aa
AT ROOM USING 1 CITl
Figure 2 shows the activity of crude bee venom PLA, and pure PLA, at high temperatures when egg yolk was used as the substrate. A linear rise in enzymic activity occurred with the increase in temperature and at 65°C the activity was maximal. Further increase in temperature resulted in a sudden decrease in activity to cu. 4% of the maximum activity, at 75°C. The same temperature optimum was noted when the egg yolk dispersion, which was heated to 90°C for 10 min, cooled and reblended, was used as the substrate or when hard
30
40
60
50
70
80
“C
FIG.
2.
EFFECT OF TEMPERATURE
(-+-•--);
TOXICON
1976 Vol. 14
ON THE ACTIVITY OF CRUDE ACCIVITY.
and purified bee venom PLA, activity -O-O--. dispersion.
BEE VENOM
PHOSPHOLIPASE
Substrate
AB
was egg yolk
CHITRA
38
NAIR,
JAN HERMANS,
D. MUNJAL
1
1000
40
50
and W. B. ELLIOTT
I
I
60
70
"C
FIG. 3. EFFECT OF TEMPERATURE ON PIIRIFIEDBEE VENOM PHOSPHOLIPASE A, ACTIVITY USING SONICATEDDIPALMITOYL LECITHIN,(--0--0-) AND DICAPROYL LECITHIN(-.-.--).
boiled egg yolks were dispersed instead of raw egg yolk. However, at 80°C the enzyme showed about 2% of the maximum activity with the addition of a highly concentrated solution of PLA,. The activity of pure bee venom PLA, with both sonicated dipalmitoyl lecithin and dicaproyl lecithin showed a maximum activity at 60°C in both the cases (Fig. 3). The activity of PLA, (egg yolk substrate) was totally suppressed by the addition of 10 ul of specific antiserum (rabbit) to the enzyme (prepared by immunization with pure enzyme) and incubating the mixture for 15 min at 37°C. Serum, up to 100 pl, from an
ii,
, 64
68
/
,
72
76
, 8.0
PH FIG. 4. EFFECTOF~H
TOXICON
1976 Vol. 14
AT~~~CONCRUDEBEEVENOMPHOSPHOLIPASEA~ACTIVITY. Substratewas egg yolk dispersion.
Temperature
Stability of Phospholipase-I
39
unimmunized rabbit did not inhibit the activity. This observation indicates that the measured activity was due to PLA, only. As shown in Fig. 4, the pH profile of the reaction at 65°C showed a maximum activity at pH 7-O as contrasted to a pH optimum of 8-O at room temperature. The effect of urea on the activity of PLA, at higher temperatures using egg yolk as substrate is shown in Fig. 5. At 40 and 50°C there is no change in the activity of PLA, with the addition of up to 2 M urea but with a higher concentration of urea a sudden loss of activity was noticed and the activity was completely lost in the presence of 4 M urea. At 60°C the activity loss was observed on addition of O-7 M urea and the enzyme became completely inactive with 2 M urea. Table 1 shows the effect of ethanol on the activity of phospholipase A, at 30 and 40°C. At 3O”C, an appreciable increase in activity was observed with lo,20 and 30% ethanol concentration but the activity became 0 at 40% ethanol. The activity was almost doubled with 10 and 20% ethanol at 40°C but was reduced with 30%
FIG. 5. EFFECTOF UREAON PUREBEEVENOMPHOSPHOLIPASE A, ACTIVITY. 0 4O”C, A 5O”C, !J 60°C. Substrate was egg yolk dispersion.
TABLE 1. EFFECTOF ETHANOLON THE PHOSPHOLIPASE A, ACTMTY
Alcohol concentration (%) 0 10 20 30 40
pmole of fatty acid released/mg of proteinjmin 30°C 40°C 312 550 444 1190 444 1190 432 680 0 0
Substrate used was egg yolk dispersion. TOXICON
1976 Vol. 14
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CHITRA NAIR, JAN HERMANS,D. MUNJAL and W. B. ELLIOTT
ethanol and became zero at 40 ‘A. Due to the evaporation of alcohol at higher temperatures, the effect of alcoho1 on PLA, activity couId not be studied above 40°C. DISCUSSION Previous studies employing the hydroxamic acid assay with dipalmitoyl lecithin, dispersed by vortex mixing, as substrate showed the optimum temperature for bee venom PLA, activity to be 50°C (MUNJALand ELLIOTT,1972) which is in close agreement withthe work of WV and TINKER(1969) with PLA, from Crotdus atrox venom. When egg yolk was used as a substrate, PLA, showed an abrupt loss of activity above 65°C. Similarly, sudden loss of activity was observed with sonically dispersed dipalmitoyl lecithin, and dicaproyl lecithin at 60°C. This loss of activity could possibly be due to the abrupt change in the physical nature of the substrate, as the flocculation of the substrates starts at these temperatures. The above observations suggest that one of the reasons for the loss of activity could be the nonavailability of the substrate to enzyme due to a change in physical state of the substrate. However, the study on the effect of urea on the enzymic activity at elevated temperatures shows no loss of activity at 40 and 50°C in the presence of urea concentration up to 2 M, whereas at 60°C the enzyme starts losing its activity with the addition of urea to O-7 M and the activity becomes zero, at a urea concentration of 2 M. The increased sensitivity of the enzyme to urea at 60°C could indicate a weakening of a possible hydrophobic bonding critical to maintenance of the conformation of the active site. This suggests that the loss of activity at about 65°C might be due not only to the possible change in the physical nature of the substrate but could be due to a minor change(s) in conformation of the enzyme localized at or near the active site and/or the substrate recognition site. If such a change occurs on heating, it is reversible as the enzyme preheated without substrate at 90°C for 15 min at pH 8.0 folIowed by cooling also gave an optimum temperature of 65°C with egg yolk substrate. However, in the presence of cu. 1 M urea the incubation of the enzyme at 90°C for 15 min resulted in a total loss of activity. Earlier studies of heat stability of the enzyme were usually performed at pH 5.9 (HUGHES, 1935). However, the O.R.D. spectra of pure bee venom PLAz remained unchanged even at 90°C. In the presence of ethano1 at 30 and 4O”C, there was considerable change in the activity of PLA,. With 10 and 20% ethanol concentration increased activity was observed at 30 and 40°C and with 30% ethanol the reduction of activity was noticed in both the temperatures. But the decrease in activity with 30 % ethanol was greater at 40°C (ca. 45 % of the activity in 20% ethanol) than at 30°C (2%). This marked decrease in activity with a 10°C difference in temperature is quite significant, even though our results from O.R.D. studies showed no change in specific rotation in 50% ethanol at room temperature. Since the O.R.D. spectra show much less helical content for bee venom phospholipase A, than is present in porcine pancreatic phospholipase A,, it is not unexpected that the change in the biological properties of the enzyme due to reversible heat denaturation apparently did not cause any significant change in the optical rotatory dispersion of the - enzyme protein (measured in the absence of substrate). At 80°C the enzyme showed about 2 % of the maximum specific activity when measured with a highly concentrated solution of PLA, and egg yolk substrate. The catalytic activity of the enzyme is low at this temperature, and cannot be detectable with the usual low concentrations of the enzyme used in the assay at lower temperatures. TOXICON
1976 Vol. 14
Temperature
Stability of Phospholipase-I
41
The possible causes for loss of PLA, activity at elevated temperatures are : (1) a change in the substrate that makes it unavailable to the catalytic site of the enzyme and/or to the micelle recognition site of the enzymes which is necessary for maximal activity, (2) a minor localized reversible change in the conformation of the enzyme molecule which changes the catalytic activity by altering the active site but does not change the O.R.D. spectrum, (3) a loss of the micelle recognition site because of a reversible limited local conformational change, (4) an action of some other agent in the venom, on either the substrate or the PLA,, which has a very steep activation curve at high temperatures. In the case of egg yolk substrate, conceivably denaturation of the lipoprotein would render it unsuitable as a substrate; however, egg yolk substrate prepared from hard boiled egg yolks gave the same temperature optimum as did egg yolk substrate heated to 9O“C, allowed to flocculate and redispersed. The action of other factors in the venom on either the substrate or PLA, was eliminated by using pure PLA,. Since both the substrate dispersion structure and the conformation of the enzyme are stabilized by hydrophobic bonding, the experiments with urea and ethanol do not yield evidence clearly in support of any of the possibilities. However, heating the enzyme to 90°C in the absence of substrate or urea did not result in a loss of activity measured at lower temperatures, while heating to 90°C in the presence of 1 M urea resulted in a loss in activity when measured at lower temperatures, clearly indicating an action of urea on the enzyme molecule at high temperatures. At room temperatures ethanol up to 50% (v/v) produced no alteration in O.R.D. spectra; however, ethanol stimulated the hydrolysis of phospholipid. At WC, ethanol (10 and 20% v/v) was stimulating but 40% was totally inhibiting. This sharp loss in activity would seem to favor a change in the enzyme molecule as other factors affecting disperions, e.g. sonication, do not cause a total loss of activity at this temperature. Additional evidence favoring conformational change in the enzyme as the basis for the loss in activity is presented in the following paper (NAIR etal., 1976). Acknowledgement-The authors thank Mr. G. W. SHEPHERD for the purification of bee venom PLAl and Dr. JOHN WYPCH for a generous gift of specific antisera. This work was supported by a U.S.P.H.S. grant GM06241 and a fellowship to C.N. from the United Health Foundation. REFERENCES AUGUST~N,J. M. and ELLKIIT, W. B. (1969) A modified hydroxamate
assay of phospholipase A activity. Anal. Biochem. 31,246. BROWN,J. H. and BOWLES,M. E. (1966) Studies on the phospholipase A activity of Crotalus atrox venom. Toxicon 3,205. CONLIREA, E., DEVRIES,A. and MAGER,J. (1964) Hemolysis and splitting of human erythrocytes phospholipids by snake venoms. Biochim. biophys. Acta 84,60. DEHAAS, G. H., POSTEMA, N. M., NIEUWENHUIZ EN, W. and VAN DEENEN,L. L. M. (1968) Purification and properties of phospholipase A from porcine pancreas. Biochim. biophys. Acta 159,103. DESNIJELLE, P., CONSTANTIN, M. H. and BALDY, J. (1955) Technique potentiometrique pour la mesure de l’activitk de la lipas pancreatique. BUN. Sot. Chim. biol. 37,285. DITTMER,J. C. and LISTER,R. L. (1964) A simple specific spray for the detection of phospholipids on thin layer chromatograms. J. Lipid Res. 5,126. FAIRBAIRN,D. J. (1945) The phospholipase of the venom of the cotton-mouth moccasin (Agkistrodon piscivorous L.). J. biol. Chem. 157, 633. HABERMANN, E. (1957) Pharmacology of phospholipase A. Archs exp. Path. Pharmac. 230,538. HANAHAN,D. J. (1952) The enzymatic degradation of phospharidyl choline in diethyl ether. J. biol. Chem. 195, 199. HANAHAN,D. J., RODBELL,M. and TURNER,L. D. (1954) Enzymatic formation of monopalmitoleyl- and monopalmitoyl lecithin (lyso lecithins). J. biol. Chem. 206,431. HUGHES,A. (1935) The action of snake venoms on surface films. Biochem. J. 29,437. TOXICON
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KAWAUCHI,S., IWANAGA,S., SAMEI~MA,Y. and Suzurcr, T. (1971) Isolation and characterization of two phospholipase A’s from the venom of Agkistrodon halys blornhofi. Biochim. biophys. Acta 236,142. LONG, C. and PENNY, I. F. (1957) The structure of naturally occurring nhosuhonlycerides. 3. Action of mocassin-venom phospholipase A on ovolecithin and related substat.& BioEhem.J. 65, 382. MAENO,H., M~~~IJHASKI,S., OKONOGI,T., HOSHI, S. and HOMMA,M. (1962) Studies on habu snake venom. V. Myolysis caused by phospholipase A in habu snake venom. Jap. J. exp. Med. 32,55. MAGEE,W. L. and THOMPSON,R. H. S. (1960) The estimation of phospholipase A activity in aqueous systems. Biochem. J. 77, 526. MUNJAL,D. and ELLIOTT,W. B. (1971) A simple method for the isolation of a phospholipase A from honey bee (Apis mellifera) venom. Toxicon 9,403. MUNIAL, D. and ELLIOTT,W. B. (1972) Further studies on the properties of phospholipase A from honey bee (Apis mellifera) venom. Toxicon 10, 367. NAIR, B. C., NAIR, C. and ELLIO~~, W. B. (1976) Temperature stability of phospholipase A activity-II. Variations in optimum temperature of phospholipases Aa from various snake venoms. Toxicon 14, 43. ROHOLT,0. A. and SCHLAMOWITZ, M. (1961) Studies of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A. Archs Biochem. Biophys. 94,364. SCANU,A. M., VAN DEENEN,L. L. M. and DE HAAS, G. H. (1969) Optical rotatory dispersion and circular dicroism of PLA, and its zymogen from porcine pancreas. Biochim. biophys. Acta 181,471. SHEPHERD,G. W., E~~orr, W. B. and ARBESMAN,C. E. (1974) Fractionation of bee venom. I. Preparation and characterization of four antigenic components. Prep. Biochem. 4,71. Wu, T. W. and TINKER,D. 0. (1969) Phospholipase Aa from Crotalus atrox venom. I. Purification and some properties. Biochemistry 8, 1558. ZAMECNIK,P. C., BREWSTER,L. E. and LIPMAN,F. A. (1947) Manometric method for measuring the activity of the Cl. welchi lecithinase and a description of certain properties of the enzyme. J. exp. Med. 85,381.
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