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Studies of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A
ARCHIVES
OF
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
364-379 (1961)
of the Use of Dihexanoyllecithin and Other as Substrates for Phospholipase A’
With Addendum
on Aspects of Micelle 0. A. ROHOLT
From
94,
the Division
AND
Properties
of Dihexanoyllecithin
M. SCHLAMOWITZ
of Research Biochemistry, Roswell Park Memorial for Cancer Research, Buffalo, New York Received
Lecithins
December
Institute
29, 1960
Some properties of the phospholipase A from venom of Crotalus durissvs terrificus have been studied using as substrate a water-soluble lecithin, dihexanoyl-L-a-glycerylphosphorylcholine (DHL). Ca++ is required for activity, and the concentration required for maximal activation is about 0.004 M at pH 6.8-9.8. The pH optimum for the system in the presence of excess Ca++ is pH 8. Ba++, Zn++ and Cd++ inhibited the reaction. Inhibition by Ba++ was competitive with Ca++, and ‘most probably involves a site on the enzyme. Caproate, one of the products of the enzymic reaction, has essentially no effect on the reaction even at levels 10 times that of the substrate. Hexanoyllysolecithin, the other product, had no effect when t,ested at 5 X 1Om6M. The influence of other lysolecithins , sodium dodecyl sulfate, Tween 20, and Tween 80 on the activity of the enzyme toward DHL, and toward the water-insoluble dioctanoyl-, dimyristoyl-, and ovolecithin is also described. Studies on the micellar properties of DHL, with estimates of t,he critical micelle concentration and micellar weight are also presented. INTRODUCTION
of Ca++ activator are restricted by the insoluble nature of the long-chain fatty acids and their calcium soaps, and by the micellar nature of the lysolecithins. Although the difficulties arising from the use of heterogeneous or impure lecithins can be surmounted by use of purified dipalmitoleyllecithin such as has been isolated from yeast (2), the complications associated with the water-insoluble nature of the high molecular weight lecithins remain. The finding of Hanahan (3) that natural lecithins are hydrolyzed in moist ether by phospholipase A has been of great value in studies of the enzyme and has provided a useful method for the preparation of lysolecithins. However, studies on the effects of ionic strength, pH, and of substances insoluble in ether are obviously not amenable to this medium ; nor is it known with certainty that all components of this system are in simple solution. In both the aqueous emulsion and ether systems enzymic hy-
St’udies of the action of phospholipase A on lecithin are often complicated by the nature of the substrate. Many lecithins from natural sources are heterogeneous with respect to their fatty acid residues, posing problems of substrate specificity. Further, most of the lecithins are insoluble in water and necessitate the use of multiphase systems in which the degree of dispersion of substrate and its relation to enzyme activity is poorly defined. Yet another element of undetermined significance for enzyme action on lecithin emulsions is the demonstration that micelles or emulsions of natural lecithins are able to ‘(adsorb” electrostatic charges (1). It may further be noted that with the lecithins of high molecular weight, studies of the influence of the hydrolysis products (fatty acids and lysolecithin) and ‘This investigation was supported in part by a research grant (No. C2585) from the National Institutes of Health, U. S. Fublic Health Service. 364
ACTIOS
OF PHOSPHOLIPASE
drolysis often does not proceed as a linear function of time, but manifests a lag period (4, 5) of undefined etiology. In an earlier communication (6)) dihexanoyllecithin (DHL) ,2 a short-chain lecithin was investigated for possible use as a water-soluble substrate for phospholipases. It was shown that DHL can serve as substrate for phospholipase ,4 and that neither a lag phase nor precipitation of the calcium soap is encountered. The present study further characterizes this system with respect to the pH for optimal activity and the influence of activator and inhibitor metal ions and of hydrolysis products. Some comparative studies are also presented of the action of phospholipase A on some of the water-insoluble lecithins, c.g., ovolecithin, dimyristoyllecithin, and dioctanoyllecithin. Observations of the influence of surfactants like Tween 20 and lysolecit,hins are also recorded. MATERIALS
PHOSPHOLIPASE A (PL-A) The (Miami
enzyme preparation was dried venom Serpentarium) from Crotalus tlurissus ter-
rificus.
LECITHINS Synthetic dihexnnoyllecithin (DHL), dihexanoyl-L-a-glycerylphosphorylcholine (8) was obtained from Dr. Erich Baer of the Banting Institute, Toronto. Anal. Calcd. for GH,?O&P: N, 2.97; I’, 6.57. Found N, 2.92; P, 6.62. Bnalysis for rholine (9) rhor;-ed 29.7% (as choline chloride) ; theory, 29.6%. Synthetic dioctanoyllecithin (DOL) was also obtained from Dr. Barr. D;myristovllerithin (DML), synthesized acrording to Baer ct r/l. (lo), Lamotte Chemical co. Anal. Calcd. for C,,H,,O,NP: N, 2.02; P, 4.45. Found N, 2.10; P, 4.41. ‘As may be noted by comparison with a previous paper (6), the nomenclature of the lecithins llas been changed. DHL was formerly designated as DCL. The change has been made to avoid confusion that, might arise from the use of the trivial names of the, fatty acid substituents of lecithins, in the G-C,, range. The present system conforms to the nomenclature in use (7) for lecithins and rcphnlins in this group.
OK LECITHINS
365
Ovolecithin (OvoL) was prepared by the chromatographic procedure of Rhodes and Lea (11) and stored in methanol-chloroform (30:70 v/v) solution at -10”. In one of several runs the elution peak was divided into three parts. The leading portion, the central portion, and the slowest moving portion were each analyzed for phosphorus, choline, and fatty arid. The expected 1:1:2 ratio for these constituents was found. The slowest moving portion is reported lo rontain the more saturated lec,ithins (12).
Myristoyllysolecithin (MLL) was prepared from DML by the action of the ycnom enzyme by a procedure based on the methods of Hanahan rt rtl. (13), and of Saunders and Thomas (14). A solution of venom in 0.005 ‘$1 CaCl, was incubated at room tempcraturc with a solution of DML in 4Yc ethanol in ether. The insoluble lgsolccithin produced was reprccipitated from chloroform with ether until crystalline, and finally was crystallized three times from hot ethanol. Ovolysolecithin (OvoLL) ~3s prepared from ovolecithin in a similar manner using the slower portions of the elution peak. The OroLL was recrystallized five times from hot ethanol. The preparation of the OvoLL involved the use of considerably more enzyme than did the preparation of the MLL; it was still contaminated with cnzyme ercn aftrr five recrystallizations (rf. Results, Fig. 6). Marplcs and Thompson (15) working with OroLL prepared using venom from the cottonmouth moccasin were able to eliminate adsorbed enzyme by recrystallization of the Iysolecithin.
CAPROIC ACID The caproic acid used was assayed by titration. Neutral equivalent: found 119; theory 116. METHODS
Assay OF PHOSPHOLIPASE A ACTIVITY Enzymic hydrolysis of the lf,cithins was followed by continuous titration of the liberated fatty acid at, constant pH, using a Beckman model G pH meter with extension probe electrodes. The assay system was standardized to 1 ml. total volume and contained lecithin, CaC12, ethylenediaminetetraacetic acid (EDTA), NaCI, buffer, and enzyme. The concentrations of the lecithins (0.002-0.005 JI) and of Ca” (O-0.02 M) will be specified for each experiment. It is important to note that these levels of substwtr arc in the range where vrry little if any dihexanoyllccithin is present as miccllrs (WC Atldcntlrc~n). EDTA (0.00071
366
ROHOLT
AND
SCHLAMOWITZ EXPERIMENTAL
AND
RESULTS
M) was used along with excess Cat+. The activity of the enzyme was increased several fold by its presence, presumably due to binding of inhibitory ions3 The NaCl (0.1 M) was used to minimize differences in ionic strength between experiments ; and the inclusion of a small amount of buffer (5 X 10m4 M maleate, Tris, or aminomethylpropanol) made it practicable to control the pH to within 0.2 pH unit during titrations. Enzyme solutions were prepared from the dried venom in 0.00355 M EDT$, dialyzed in the cold against several changes of 0.01 M CaCh, and then centrifuged clear. They contained about 5 pg. of dry venom/pi. The reactions and titrations were carried out under nitrogen in small vials with rapid mixing. A l-ml. Gilmont ultramicroburet calibrated to 0.0001 ml. was used for the titration with 0.100 N NaOH. Under these conditions there was no CO, blank; and preliminary experiments established that nonenzymic hydrolysis of substrate in the pH range used in these studies was negligible. It was further ascertained that formation of the titratable acid was not the result of the action of proteases on the protein of the venom. Titrations leveled off at values which corresponded approximately to the liberation of one mole fatty acid per mole lecithin and they could not be increased by further addition of 20-30 times the amount of venom normally used (15-30 pg.). In all the experiments in this study, complete titration curves were recorded relating the micromoles of NaOH taken up as a function of time of hydrolysis. In those experiments where the relevant effects are best shown by the complete curves (Figs. 5-7), they are drawn. In the others where the relations of interest are the relative activities (Figs. 1-3, Table I), the activities are expressed as the reciprocal of the “time values” (17), i.e., the reciprocal of the time (minutes) to achieve 20 or 30% hydrolysis of substrate, l/T, and l/T, Thus, in a reaction employing 1 ml. of 0.002 M substrate, a value l/T, of 0.2 represents the reciprocal of the time (5 min.) for 20% (0.4 pmole) hydrolysis. The l/T,0 and l/Tm values are on the linear or near linear portions of the hydrolysis curves. Reproducibility of titration data was satisfactory, generally within 5%, but occasionally varied by up to 15% (cf. Figs. l-4).
This relationship was established in the standard system with levels of venom solution ranging from 0.2 to 6.0 ~1. For the lower levels of venom solution (0.2-1.0 ~1.) the substrate concentration was 0.002 M DHL. For the higher ones the concentration of DHL was 0.005 M. In all cases the Ca++ concentration was 0.02 M and the pH was 8 (Tris buffer). The results, Fig. 3, show a linear relationship between activity and enzyme concentration over the entire range of enzyme concentration tested.
3 Large amounts of Zn++ has been reported in the native venom of several species of snakes (16). It is possible that the need for EDTA in these studies is related to this fact.
‘The Ca++ concentrations are corrected for the 0.71 pmole presumably bound by the EDTA present. The amount bound by the enzyme is considered to be negligible.
1. CHARACTERISTICS OF THE DIHEXANOYL-
LECITHIN-PHOSPHOLIPASE
Effect of Ca++
A SYSTEM
on the Activity
of PL-A
The enzyme from Crotalus durissus terrifkus venom was inactive in the standard system unless Ca+ +, in excess of the EDTA, was added. The influence of Ca++ was investigated at several pH levels with 5 ~1. enzyme solution and 0.005 M DHL. The buffers were maleate at pH 6.8, Tris at pH 7.8 and 8.8, and aminomethylpropanol at pH 9.8. The results, Fig. 1, show that over a wide pH range, maximal activity is achieved with 0.004 M Ca++ and that further increases in Ca++ up to 0.02 M are without further effect4 Optimal
pH for the Activity
of PL-A
A pa-activity curve was then obtained with the standard assay system containing 0.002 M DHL, 0.02 M Ca+ +, and 3 ~1. of venom solution. From pH 6 to 7, maleate buffer was used; Tris from pH 7 to 8.8, and from pH 9 to 10 no buffer was necessary. The curve, Fig. 2, is symmetrical with a broad maximal region centering around pH 8. Relation
between Enzyme Concentration and Rate of Hydrolysis
ACTIOh’
OF PHOSPHOLIPASE
367
ON LECITHIKS
pH 9.9
FIG. 1. Activation
Temperature
of phospholipase
Coeficient
A by Ca++ at several
pH values. See text for details.
of Activity
The standard system was run at 20, 30, and 40” at pH 8.0 (Tris buffer) with 0.005 Ilf DHL, 0.005 ,I[ Ca+ +, and 3 ~1. venom. The temperature coefficient of the activity ( l/Tso) per 10” interval was 1.55 and 1.45 for the 20-30” and 30-40” intervals, respectively, and the energy of activation calculated from the Arrhenius equation was 7300 cal./mole for the hydrolysis of DHL by this enzyme. Influence of Some Metnl Ions on the Activity of PI,-A The effect of Mg++, Ba++, Zn++, Cd++, &In++, Co++, and Cu++ was tested at several concentrations and pH values in the standard system with 0.005 M DHL and 0.005 M Caf f. This level of Ca+ + (only slightly in excess of the minimum re-
FIG. 2. Activity of phospholipase noyllecithin as a function of pH.
-4 on dihexa-
quired to give maximal activation, cf. Fig. 1) was selected to permit effects of the other cations to be manifest. None of the ions tested increased the activity beyond the level already achieved by the Ca+ -1.present. At pH 8, Mg++ (0.005,
368
ROHOLT
ENZYME
AND
SCHL.4MOWITZ
CONCENTRATION
( x venom solution)
FIG. 3. Rate of hydrolysis of dihexanoyllecithin as a function of enzyme concentration. Activity values on the two plots are not comparable since the substrate concentrations are different. See text for detail:
0.02, and 0.05 M) did not affect the reaction rate. Mn++ (0.005 M) did not inhibit but gave a small precipitate even when the pH was lowered to 6.8. Co++ (0.001 and 0.005 M) and Cu++ (0.005 M) were inhibitory (50-90%) at pH 6.8 but were not amenable for further study because they too gave precipitates at this pH. Ba++ at pH 8 was found to inhibit the reaction without forming any precipitates and was thus suitable for a study of the kinetics and mechanism of its inhibitory action. If the inhibition is the result of a competitive relation between Ba++ and Ca++, it will be determined by the (Ba++) : (Cat-+) ratio rather than by the absolute concentrations of the ions. Activity at pH 8 was measured at 0.005 and 0.002 M Ca+ + for several concentrations of Ba++. The concentration of DHL was 0.005 M, and 3 ~1. of venom solution was used. A plot of activity versus (Ba++) : (Ca++) ratio, Fig. 4 curve (a), shows that the degree of inhibition depends on the (Ba++) : (Ca++) ratio and is independent’ of the individual ion concentrations. Verification of this competitive relationship is also seen in the straight line obtained in the plot l/w versus (Ba++): (Ca++) in Fig. 4 curve (b). *4 study of the inhibitory action of Zn++
at pH 8 was ruled out by the formation of a heavy precipitate. At pH 6.8, however, no precipitate formed. At this pH the reaction could be inhibited 95% by 0.00085 M Zn+ +5 and released by addition of sufficient EDTA to bind the Zn+ +. In this experiment, Fig. 5 curve (a), 5 ~1. of venom solution was added to 0.72 ml. of a solution containing 5 pmoles DHL, 0.71 rmole EDTA, 5 pmoles Ca+ +, NaCl, maleate buffer, and 0.85 pmole Zn+ +. Under these conditions the reaction was inhibited by the Zn++. After 8 min. 0.27 ml. of a solution containing 0.6 pmole Ca++ and 0.71 pmole EDTAG adjusted to pH 6.8, was added. The total amount of EDTA present (1.42 pmoles) exceeded the amount of Zn++ (0.85 pmole) and, because of its great avidity for the metal, reduced the level of free Zn++ to a noninhibitory level. The enzymic reaction then proceeded at about the control rate [compare with curve (b)]. A second quantity of Zn++ (0.85 pmole) in a volume of 0.017 ml. was added 5 min. later. Again the total amount of Zn++ (1.7 pmoles) slightly exceeded the total amount of EDTA (1.42 pmoles) , and again the enzymic reaction essentially stopped. After 3 min., a second portion of Ca-EDTA solution was added, and as before the enzymic hydrolysis resumed at about the same rate as the control.
‘The stability constants for Zn-EDTA and Ca-EDTA are approximately 10” and lOlo, respectively (18). Since the reaction mixture contains 0.00071 M EDTA which binds the Zn++ tightly, the actual concentration of Zn++ is closer to 0.0001 M.
‘EDTA was added as the Ca chelate (pH 6.8) to avoid the liberation of H+ (0.7 wmole) which would have resulted if the NasH (EDTA) salt were used to chelate the Zn++. The additional trace amount of Ca++ contributes little to the already maximally stimulating concentration present.
ACTION
OF PHOSPHOLIPASE
ON LECITHIM
369
- 25
1 5
FIG. 4. Relation between t,he ratio of [Ba”] : [Ca-+] and the activity of phospholipaae Curve (a), activity versus [Ba++]:[Ca++]; curve (b), l/v versus [Ba‘*]:[Ca++]. O-0, o-e, 0.005 M ca+-; U---O, B-I, 0 02 31 Cat+. See text for detail.
TIME
A.
(minutes)
FIG. 5. Effect of alternate additions of inhibitor ions (Zn++ or Cd-‘) and of EDTA on the rate of hydrolysis of dihexanoyllecithin by phospholipase B in the presence of Cat+. Curve (a), inhibitor ion, Zn”; curve (b), control for curve (a). Curve (c), inhibitor ion, Cd++; curve (d) control for curve (cl. See text for details on amounts and order of additions of inhibitor ions and EDT$.
Similar experiments were carried out at pH 8 with Cd+ ii using 3 pl. venom. Though ‘The sentially
stability constant for Cd-EDTA the same as for Zn-EDTA (18)
is es-
somewhat less effective than the inhibition produced at pH 6.8 by Zn++, inhibition by Cd+ + at pH 8, Fig. 5, curves (c) and (dj , could also be counteracted by EDTA.
370
ROHOLT
AND
SCHLAMOWITZ
TIME
(minutes)
2.ot-
1.8 0.0 1.6 0 I .4
0 n .-----
M OVOLL.
5Xlo+M
OVOLL.
3.3Xl0‘4M
OVOLL.
3xto-3rd OVOLL. 8XIO-3M OVOLL. 5X10- M MLL.
1.2 i 2
1.0
i 32 0.8 E 25
0.6
FIG. 6. Comparisons of the enzymic hydrolysis ovolvsolecithin and in its absence (0.0 M OvoLL). is also shown.
Influence of Caproate on the Activity of PL-A The effect of caproate, a product of the action of PL-A on dihexanoyllecithin was determined in the standard assay system with 3 ~1. of venom solution, 0.005 M DHL, and 0.02 M Ca++ at pH 8 (Tris buffer). At concentrations of 0.001, 0.01, and 0.05 M corresponding to 0.2, 2, and 10 times the initial DHL concentration, caproate caused less than 10% inhibition of the reaction rate. In,fluence of Lysolecithins on the Hydrolysis of Dihexanoyllecithin by PL-A Figure 6 depicts the hydrolysis of DHL in the standard assay system containing 0.002 M DHL, 0.02 M Ca++, and 3 ~1. venom solution at pH 8 (Tris buffer) in the presence of ovolysolecithin (1 x 10e5 to
of dihexanoyllecithin in the presence of The effect of myristoyllysolecithin (MLL)
8 x low3 M)s and in its absence (0.0 J!f OvoLL). The Ca+f was added 10 min. prior to the addition of the enzyme. It is seen that compared with hydrolysis in absence of OvoLL, hydrolysis of DHL is increased roughly 3-7-fold by the presence of OvoLL. The significant ‘Lblank” during the preincuba,tion period, due to residual enzyme activity in the OvoLL (see Materids), seen at the two higher levels of OvoLL (0.008 and 0.003 M) becomes negligible at lower levels of OvoLL. The possibility that the increased hydrolysis of DHL by the presence of OvoLL might be due to the presence of some naturally occurring stimulatory substance in the OvoLL was ruled out by a comparison of *Solutions of lysolecithins were used within 24 hr. since it was observed with both MLL and OvoLL that precipitates formed after 2 or 3 days at room temperature.
ACTION
OF PHOSPHOLIPASE
these results with the ones obtained with myristoyllysolecithin enzymically prepared in ether from synthetic dimyristoyllecithin (Uoterials). At 5 x 10-j M it is seen that MLL and OvoLL produced comparable effects (Fig. 6). To further insure that this property of both OvoLL and MLL was not in any way related to their preparation in the ether system, a sample of MLL was prepared by the action of the enzyme on DML in an aqueous system and tested as follows: a suspension containing 10 pmoles DML in 0.01 iM Ca++ at pH 8 (Tris) and 4 ~1. venom solution was incubated according to the standard assay procedure for a period of 160 min. (94% hydrolysis). Portions of this reaction mixture (0.06,0.006, and 0.002 ml.) sufficient to produce final concentrations of 5 x 10-4, 5 x 10-j, and 1.7 x lo-” M MLL in the standard procedure were t,hen tested using 0.002 M DHL substrate, 0.02 M Ca++, and 3~1. venom solution at pH 7.8 (Tris buffer). The MLL prepared in this manner stimulated the reaction the same as did the MLL and OvoLL prepared by the ether system. Hexanoyllysolecithin, the normal product of reaction of PL-A on DHL, prepared in water as was the MLL, had no effect at a level of 5 X 10Ws M. It is seen from these studies that lysolecithins derived from synthetic DML and from OvoLL greatly increase the activity of Crotalus durissus terrificus venom PL-A toward DHL. A similar effect of OvoLL on the activity of purified bee venom PL-A toward OvoLL was attributed by Habermann (4) to finer emulsification of the substratc. It, is unlikely that such an interpretation applies in the present study since the dispersion of DHL at the concentration used here is essentially complete (see Adden&m). A satisfactory explanation for the above observations is not readily apparent. Influence of Sodium Dodecyl Sulfate (SDS), Tween 80 and Tween 20 on the Hydrolysis of Dihexnnoyllecithin by PI,-A Inasmuch as lysolecithins are surfacc-actire agents in addition to having structures related to the substrates for PL-A, ot’her
ON LECITHINS
371
surface-active agents were investigated for possible stimulatory action. The substances were test,ed in the standard assay procedure using 0.002 M DHL and 0.02 M Ca++ at pH 8 (Tris buffer). The conditions used and the results are given in Table I. In the presence of SDS, (0.0026 or 0.037 111) hydrolysis was completely inhibited when as much as 500 ~1. venom solution were used. Tween 80 at 0.26% and Tween 20 at 0.038 and 0.75%, had little effect on the hydrolysis. At 3.8%, Tween 20 inhibited the reaction about 46%. This inhibition was not altered or reversed by the presence of 5 x 109” J!1 JILL. Action of PL-A on Dihexanoyllecithin 4% Ethanol-Ether Solution
in
This experiment was carried out to establish whether DHL could be hydrolyzed by PL-A in the ether system the same as were the higher molecular weight lecithins. Venom (10, 50, and 100 pg. in 0.01 ml. of 0.01 M CaC12) was added to samples (1 ml.) of the ethanol-ether subst’rate solution (2 ~molcs DHL) , and the reaction was followed at, room temperature by titrations of the mixture according to Hanahan et al. (13). The reactions proceeded without any lag period. Relat,ive initial rates of 1.0, 0.5, and 0.03, respectively, were observed for 100, 50, and 10 pg. venom, and hydrolysis proceeded to about 8076 of theoret’ical. 2. COMPARATIVE EFFECTS OF MYRISTOTLLYSOLECITHIN.~ND OTHER SVRFACE-ACTIVE COMPOUA-DSONTHE A4c~rvr~r OF PHOSPHOLIPASW,A TOWBRD DIOCT~~NOYL-, DIM~RTSTOYL- U-D OVOLECITHIN
The following experiments describe t,he influence of myristoyllysolecithin (MLL) , sodium dodecyl sulfate, and Tween 20 on the action of PL-A on some of the waterinsoluble lecithins. Influence of MLL on the Hydrolysis T)OL, DML and Oz~oL by PL-A
of
The effects of MLL mere tested by the standard assay procedure with 0.002 Jll of the appropriate lecithin and 0.02 ,W Ca++ at pH 8 (Tris buffer). Several concent,rations of JILL and of v(lnom were used, and
l)Ol. Dhll, OVOL
Twain
20 3.8’ ;, :1.cv:; 3.8“(
0.03b 3” 3
0.x r 0.0
0.05 c 0.22
85 _
* For the control w-here the rate \VAS too fast to measure, the value 27 was ot)t:Cnecl hy cdculation. 500/S x 0.16. for 5 or 90 niin. or 20 hr. at 5” did not b I’sposure of venom to 3.8’,&* Twecn 20 :tt room 1emper:iturt:s wlwn assayed against I)IIl.. There :we :tppnrent Iy no irrrversihle significantly change the activity chnnges of the enzyme in 3.8’,‘, Twcn 20. c SIX I:ig. 7 for comparison of control and cspcrimcntnl rates.
the rcwlts obtnintd are suuuuarizcd in Table II together with thaw previously obtained for hydroly& of L)HL. Scvcrsl p0int.s niny bc noted which focus attention on the colliplcxity of thrx various systmis. i\ltt~ougti Yolubility of I)Hl, is aplxmntly not n factor, its rntc of hydrolysis is incrcawl scvcml fold in ttw t)rcwirc of NLL. On tlw ottwr hnntt, ciuulsiona of INI, arc cou~tktcly clnrific>tt by 2.3 X IO-: -11 JILL. but its ratr of hydrolysis is not, nffcctcd. \Yith I>JIL as it substratc~. tticro is no visiI)lc clffwt on the solubility at the lower conwntrations of 11I.T., but ttw lag twiod in ttw tly(lrotysis is rcduwd. Xt ttw highwt concwitrntion of JII,I. the D.\IT, is ahno& couipl~~tcl~ solubilizcd and the tag is climiriatcd. In the cab0 of 0~01,. cwlntially no hydrolysis owurs in tlic abetmcc of MI,I,. In its t)rcwncc~ hydrolysis ia incoulptctcF but tmth thcl r:lk and the mtmt of hydrolysis arc’ incrcawd with incrwsing amounts of JILL, and no lag period i:: seen. .L\t. 5.6 X IO-” M 1lI,I,, hydrolysis of 0~01, falls off txpidt\- :iftcr BOY wuiptrtion. but can bc
drivcu to c~ou~plction if 85 pl. \-morn solution txtlw than 3 pl. is used. The Z,~jlrmce of Tuwn
“0 on the llydroly-
TIIO level of Twecn 20 (3.8%) which C:LIIS:(Y~ about. 469;; inhibition of hydrolysis of DHL has bwn reported to almost wint)lctcly clarify suspensions of ovolccit.tiin (,4j. A comparkon of its cffccts on souio of tile othrr lccittiins was invcstigatcd. Town 20 (3.8“; ) t)rougtit about almost complete clarification of emulsions of I)OL (0.002 JZj~ DMI,, and 0~01~. The conq~lcx nature of thaw systmis tiowcvor is again Aown by the: variety of c1ffect.sseen for the reaction rat.es. With 1101, the reaction rate war; inhibited 83% (Table I). \\Yth DJlI,. that tlydrolysk rate was virtually unchanged Imt tlw lag pcriotl was eliminated I Fig. 7j. \\‘ith OvoI,, ttic hydrolysis rntc was iricwnsrd from the zero lev(ll of the control t,o n value c*omparablc to that given by hytlrolyk of DHI, (,Tnblo I 1.
ACTION
OF PHOSPHOLIPBSE
ON LECITHINS
373
TABLE II INFLUENCE OF MYRISTOYLLYSOLECITHIS ON THE HYDROLYSIS OF DIHEXANOYLLECITHIN, DIOCTANOYLLECITHIN, DIMYRISTOYLLECITHIN AND OVOLECITHIN BY PHOSPHOLIPASE A Substrate
Venom solution
0.002Y
A4LL
d.
M
DHL DOL*
3 0.03
5 x 10--b 2.3 x 10-a
DMLc
0.03
3 x 10-E
DML
0.03
DML
3
5 x 10-j
DMI,
3
1.7 x 10-a
OvoLe OVOL OVOL
3 3 3
5 x 10-b 8 x 10-d 5.6 x 10-s
OVOL
85
5.6 x 10-S
10 x 10-j
Comments” Increase in rate (see Fig. 6). Emulsion solubilized, but neither rate nor extent of hydrolysis influenced. 15.min. lag, slow reaction, hydrolysis essentially stops at about 50Gj, of completion.d 5-min. lag, slow reaction, hydrolysis essentially stops at about 50y0 of complet,ion.d Lag period short,ened, but no effect on subsequent rate or solubility. Lag period eliminated but no effect on subsequent rate; reaction mixture opalescent instead of unstable emulsion. Same as control; almost no action. Reaction rapid up to 25% hydrolysis, then stops. Solution clear, reaction rapid to GO?&hydrolysis, then very slow. Solution clear, 90% hydrolysis in 5 min., complete in 10 min.
a Compared to controls, in absence of myristoyllysolecithin. * In controls, DOL forms an emulsion when shaken in water, but separates readily into two layers. It is nevertheless attacked smoothly and about 70 times more rapidly than is DHL. The rate remains nearly constant until about 75% hydrolysis is achieved, after which the rate falls off slowly. At the completion of hydrolysis, the emulsion has disappeared and the reaction mixture is clear. c DML is not soluble but forms a suspension. Hydrolysis is preceded by a lag period. For a typical control curve, see Fig. 7. The rather abrupt increase in rate is presumably associated with the formation of some critical level of MLL, a product of the reaction, since, as seen above, the addition of 1.7 X 10m3 M MLL eliminates this lag period. d With 0.03 ~1. venom solution no hydrolysis is observed over a 30.min. period in the absence of MLL. 8 The ovolecithin forms a coarse suspension. The control rate is very slow; only about 57, (0.1 rmole) is split in 30 min. Although OvoLL, a product of its hydrolysis, activates the DHL system (Fig. 6), there does not seem to be any effect on the hydrolysis of OvoL by the OvoLL formed during the reaction here. DISCUSSION
Most lecithins isolated by lipid extraction procedures from natural sources are insoluble in water and contain saturated and unsaturated fatty acid residues of 16 or more carbon atoms, although phospholipids of bovine muscle and brain have been reported to contain small amounts of volatile fatty acid residues (19). It is thus conceivable that water-soluble synthetic lecithins like dihexanoyllecithin are model substrates for phospholipase-A in only a limited sense. But it is equally conceivable that even with the higher molecular weight lecithins, only that minute amount soluble in water and in equilibrium with t’he insoluble phase serves
as a substrate. In any event, the practical difficulties attending the use of the waterinsoluble lecithins (see I~~troductian) recommends the use and more extensive inwater-soluble vestigation of simplified model compounds like dihexanoyllecithin for studies of the mechanism and kinetics of lecithinasc action. It is seen, for example, from the hydrolysis curves of DHL and DML (Figs. 6 and 7)) that hydrolysis of the former proceeds without the complication of a lag period. As mentioned earlier, lag periods and failure to demonstrate linear relationships bctween enzyme concentration and rates of hydrolysis have been observed’> with high
ROHOLT
TIME
AND SCHLAMOWITZ
(minutes)
FIG. 7. Effect of Tween 20 (3.8%) on the hydrolysis of dimyristoyllecithin by phospholipase A (3 ~1. venom solution). O----O, control; X.---X, Tween 20. The curve obtained in the presence of Tween 20 has been displaced 8% min. to the right (Iower abscissa) to facilitate comparison of the steep portions of the two curves.
molecular weight natural lecithins as well (4,5). The pa-activity curve for DHL is symmetrical; and the optimum pH value, pH 8, would appear to be governed by the ionieation of as yet unidentified groups in the enzyme since the ionization of both the phosphate and choline groups of the substrate do not change appreciably in this pH range. An optimal value of pH 8.3 has been reported (4) for the enzyme from bee venom acting upon egg yolk. It was not established for that system whether the marked asymmet,ry of the curve and rapid fall in activity on t,he alkaline side of the optimum pH value was a cha,racteristic of the particular enzyme, or due to the state of emulsion of substrate as a function of pH, or to a reduction of free Ca*+ with increasing pH. The optimal pH for the activity of the phospholipase A from black tiger (Note&s
scu,tatus) venom is reported by Hughes (20) t’o be pH 7.8. In regard to the known (5, 21) requirement of Ca++ for activation of phospholipase A, the inhibition by Ba++ indicat’es that activation by Ca++ involves a site for which the two ions are able to compete (Fig. 4). This competitive relation may be satisfied by either of two mechanisms. In the first case the action of Ba++ is viewed as competing with Cat+ for the formation of an active Cat+-enzyme complex which catalyzes hydrolysis of the substrate. The second mechanism postulates the formation of a substrate-enzyme complex which is activated by Caf + but not by Ba++. For the first case E + Ca++ @ ECa (active); (E) (Ca++)/(ECa)
= K,
(1)
ACTION
ECa + S *
OF PHOSPHOLIPASE
ECaS + ECa + P; (ECa) (S)/(ECaS)
E + Ba++ +
= K,
(2)
EBa (inactive);
(E) (Ba++)/(EBa)
= Kb
(:3)
and the equation for conservation of enzyme is E tc,tnl= Efree + ECa + ECaS + EBa.
(4)
Efrer is essentially zero since the activity is the same at 0.005 and 0.02 M Caf+ (Fig. 1). If the remaining forms of the enzyme arc expressed in terms of ECaS, the active one, and substituted into Eq. (4), one obtams E total o=(6)+
KS
(Ba++)K,K, 1 + (Ca++)Kb(S)
(5)
Substitution of Vrn for hEtotal and II for k (ECaS) , in Eq. (5 ) and rearrangement of terms gives 1 11
(Ba++)K,K, (Ca++)Kb(S)V,
as the kinetic expression for the first mechanism. In an analogous manner, the second mechanism leads to 1 -= V
(Ba++)K,. (Ca++)KbV,
’
1 fi
(7)
A derivation based on both cases operating simultaneously also leads to Eq. (7). According to either Eq. (6) or (7)) the reaction is competitive for an “active” form of the enzyme, and a plot of l/v versus (Ba ++)/(Ca+ +), should give a straight line.g This was found to be the case [Fig. 4, curve (b)]. The data tend to rule out the likelihood that the actual substrate is a Ca-DHL complex and that Ca++ and Ba++ compete for the DHL. Under these conditions free Ca++, Ba++, and DHL would no longer be independently variable, and a plot of l/v versus (Ba++)/(Ca++j would not be linear. ’ The stoichiometric concentrations are used inasmuch as only small amount~s of the total Ba” and Ca’+ could he bound hy the small amounts of enzyme used.
OS LECITHINS
375
The very low levels of Zn++ or Cd++ capable of producing inhibition also point to primary involvement of a site on the enzyme rather than the substrate. If the relative binding of these two ions and of Ca++ by the enzyme differs by a factor of about lo6 such as is the case for their relative binding by EDTA, it would be clear why low levels of Zn++ and Cd++ inhibit the enzyme even in the presence of 50-fold levels of Ca+ + and why a demonstration of competitive inhibition at this st,age of purity of enzyme would be difficult. Such studies must await further purification of the enzymes. Yet another mechanism for apparent Ca++ activat,ion was considered by Long and Penny (5), i.e., that the fatty acids liberated by hydrolysis of lecithins are inhibitory and that Ca++ acts by combining with them. They showed such a role for Ca++ to be unlikely at least in the ether system inasmuch as maximal activation was achieved with Ca++ to substrate molar ratios of less than 1: 10, and, further, the fatty acids remained in the ether supernatant whereas the Ca+ + was carried down with the lysolecithin. This mechanism is equally improbable for the aqueous system using dihexanoyllecithin for several reasons. First, caproate, the fat,ty acid liberated by hydrolysis, exerted no inhibitory influence even at levels 10 times that of the substrate. Secondly, such a role for Ca++ could have been fulfilled by several of the other ions tested ; yet,, this was not’ the case. They were either inert or inhibitory, even in the presence of othcrwiee adequate levels of C:b+ +. Tho pattern of effects for the action of lysolecithins, sodium dodecyl sulfate, and Tween 20 on dihexanoyllccithin and on some of the insoluble lecithins is complex, and a clarification may have to await elucidation of the mechanism of action of the phospholipases and of t,he micellar properties of some of these surfactants and other lecithins. ADDENDUM
ASPECTS OF MICELLE PROPERTIES OF DIHEXANOYLLECITHIN Although consideration
DHL gave clear solutions in water, of its lipid structure raised the ques-
376
ROHOLT
ilxD
SCHLAMOWITZ
DHL (moles per liter)
FIG. 8. Relation
of DHL
concentration
to difference
in refractive
index between
solvent
and solution. tion of whether it might be present in micelle form. This point is relevant to the study on the action of phospholipase since the use of a substrate under conditions where part or all of it exists as micelles introduces new variables, e.g., the number and concentration of substrate species, and their susceptibility to enzyme action. Studies of refractive index increments versus concentration of solute, and of the diffusion patterns of DHL, both indicated that micelle formation occurs, but that under the conditions used the critical micelle concentration range, CMC,‘” is at about 0.011 M DHL. The concentrations used for the phospholipase experiments were 0.002-0.005 &f.
Relation
of Concentration of DHL to Refractive Index
For micelle-forming systems a change in the refractive index increment is observed at the CMC when this parameter is plotted as a function of solute concentration (25). The relative refractive index of solutions of DHL in an aqueous solution, pH 8, containing 0.02 M CaCl,, 0.01 J4 KaCl, 0.0007 M EDTA, 0.0005 J4 Tris, and 0.0125 &I NaOH was measured at 25” in a Brice-Phoenix differential refractometer, model BP-1000-V, at 436 mp. The instrument is sensitive to refractive index differences, in, of approximately 3 X 10.‘.
The data relating DHL concentration to An, shown in Fig. 8 was fitted best by two straight lines. Below 0.012 M the method of least squares gave a line passing through the origin with a slope of 0.0637. Above 0.012 kf a line with slope 0.0605 and intercept 38 X 10.” was obtained. The intersection of these two lines at 0.011 M represents the CMC range (25). The difference of about 5% in the slopes of the lines is similar to the values of 3.2-7.6% calculated from data of Klevens [Table I in Ref. (25)] for other micelle systems.
Diffusion
Studies with DHL
Diffusion experiments on solutions of DHL at concentrations above and below 0.011 M provided additional information on micelle formation and micelle-monomer relationships. These were carried out using a synthetic boundary cell in the Spinco model E ultracentrifuge at 19.5-21” with a 0.2-0.8” lo The narrow range of solute concentration in which micelle formation begins has been designated as the critical micelle concentration (CMC). Above this concentration both micelles and monomers exist and the dependence of many properties, e.g., osmotic pressure, conductivity, refractive index (22-24), on solute concentration is different from that observed below the CMC.
ACTION
OF PHOSPHOLIPASE
range in any given experiment, and photographs were taken at 4-min. intervals for 72 minutes. Figure 9, (a) and (b), shows representative patterns obtained when solutions of DHL at conc.entrations (0.0233 and 0.0339 ill) exceeding the CMC range indicated by the previous experiment were allowed to diffuse into the salt-buffer solvent. In both cases, a slowly spreading peak with a more rapidly spreading shoulder on the solvent aide of the peak was observed. It was found from the ratio of the area under the shoulder to the total area (obtained by drawing a vertical line at the shoulder-peak junction on all patterns) and from the t,otal conccntration of DHL, that the material represented by the shoulder at both concenirntions was equiralent to 0.011 A4 DHL (0.0103-0.0113 A4 for 0.0233 121 DHL; 0.0104-0.0120 M for 0.0339 A!1 DHL).‘l This corresponds to the value of the CMC range obtained independently from the refractjive index study. The constancy of this value at both concentrations of DHL is incompatible with the diffllsion behavior for a mixture of independent species. Although a theoretical treatment of diffusion for nonindependent species is not available for rigorous testing, the shoulder effect and the independence of the estimated CMC on concentration suggests the operat,ion of a system in which tlrrre is a rapid equilibrium bet\veen “monomerP and micelles, and in which micelles form above :I given concentration range, the CMC. The concentration gradient of the more rapidly diffusing “monomer” species is represented by the shoulder and, at levels greater than the equilibrium conrentration, the rnicelle gradient is shown by the peak. The vertical line referred to above marks this “monomerJ’-micelle equilibriunl concentration within the over-all DHL gradient in the spreading boundary. Based on this interpretation and the quantitative ~.aluc of the CMC obtained by the two methods, it was anticipated and found that diffusion of DHL at a concentration below the CMC range (0.011 .LI) yicldcd only a single rapidly spreading symmetrical “monomer” peak, Fig. 9(c) ; and that when a boundary was established between two different DHL concentrations, both greater than the CMC so that the “monomer” concentration would bc the same on both sides, only a micelle concentration gradient giving rise to a single slowly spreading symmetrical peak was seen, Fig. 9(d). ‘I No correction was made for the change of &/AC at, the CMC. This change is only about 5%. ‘* “Monomer” will be used to designate (DHL),, where for the predominant spcdies, n equals 1.
ON LECITHIKS
FIG. 9. Diffusion
patterns
of DHL.
(n) and (b), DHL at concentrations grcatel than thr CMC; 40 min. after boundary formation ; 20,410 r.p.m.; phase Illate angle, 55”. (c), DHL at a c~on~entration less than the CMC; 44 min.; 16,200 r.p.m.; phase plate angle, 40”. (d). diffusion across boundary formed by two solutions of DHL, both at concentrations grratol than !hc CMC; 44 min.: 20,410 r.p.m.; phase plate ang!c, 55”. Srfs text for intcrprclation. Diffusion coefflcicnts for the “monomers” and micselles were calculated from the data represented by Figs. 9, (c) and (d). For the micelles. values of 1.05 X IO-” and 1.06 X 10.’ s:,. cm./sec. were obtained by the “maximum ordinal e-arca” and methods (26), respectively. “maximum ordinate” For the “monomers” the> values from the average for three runs were 3.67 5 0.09 X 10.” and 3.66 -C 0.17 X IO-” scl. cni./se?.
Control
Diffusion Studies with Sucrose and Potnssiurn Law-ate
The lirnited the use of the
availability of DHL neressitated ultracentrifuge for the diffusion
378
ROHOLT
AND
studies though other systems not ‘involving centrifugal fields would have been preferred. To test the essential reliability of the results and to rule out technical artifacts, e.g., convection during synthetic boundary formation and boundary sharpening during runs even at the low speeds used, two control substances were investigated : sucrose, a simple substance with well defined diffusion characteristics; and potassium laurate, a micellar substance of defined CMC (25) not too different from the CMC of DHL. A single symmetrical peak was observed when a 0.78% aqueous solution of sucrose was allowed to diffuse into water for 44 min. Values of 5.28 X 10-O and 5.21 X 10.” sq. cm./sec. were obtained for the diffusion coefficient by the “maximum ordinate-area” and “maximum ordinate” methods, corrected to 25”. This is in good agreement with 5.20 X 10-O sq. cm./sec., the value for a 0.75% solution of sucrose at 24.95” obtained by extrapolation of the data of Costing and Morris (27). With potassium laurate (0.0555 &l diffusing into water) a shoulder was seen on the solvent side of the boundary such as was seen with DHL [cf. Fig. 9(a) and (b)]. The area of the shoulder comprised 45 f 1% of the total, equivalent to a potassium laurate concentration of 0.025 M. A similar pattern and a value of 0.025 M for the shoulder were obtained when the diffusion of a 0.048 M solution was carried out in the elect,rophoresis cell at 26”. This value of 0.025 M for the CMC range of potassium laurate obtained by the area analysis under both these conditions agrees with the reported CMC, 0.0255 M obtained by Klevens (25) by the refractive index versus concentration method, in which convection, formation of boundaries, and diffusion itself do not enter.
Sedimentation
Studies with DHL
A sedimentation run of 40 min. duration, carried out in the synthetic boundary cell at 59,780 r.p.m. on DHL at 0.005 M, a concentration below the CMC, showed only a rapid spreading of the boundary but no sedimentation. On the other hand, when a micelle concentration gradient was established by overlaying 0.0339 M DHL with 0.0170 M DHL (see Difksion Studies) sedimentation was demonstrated at 59,780 r.p.m., and a sedimentation coefficient of 0.83 S was calculated. Estimates of the maximal molecular weight for the micelle and “monomer” species of DHL may be obtained from the ralucs of the diffusion coefficients (281, assuming as a first approximation the Stokes-Einstein equation for rigid spherical particles, D = RT/GqrN, and the relation betpeen the radius, r, of the sphere and its molecular weight, M; T = (3Mp/4rN)1’S. The viscosity of
SCHLAMOWITZ the medium was taken as 0.010 poise, the value for water at 20”, and the values 6.02 X lo”, 8.3 X 10’ ergs/deg. and 293” Absolute, were used for N, R, and I’ respectively. For 8, the partial specific volume, a value of 0.91 ml/g. was used, assuming the density of solid DHL to be the same as OvoLL, 1.095 g/ml. (29). From these data a “monomer” weight of 560 and micellar weight of 24,000 were calculated. The micellar weight calculated from the sedimentation and diffusion coefficients (30) was found to be 21,400. These data indicate that there are about 45-50 DHL molecules per micelle. Considering that DHL may not be either rigid or spherical, the value of 560 for the “monomers” is in fair agreement with the formula weight of DHL, 472. At concentrations even further removed from the CMC, 0.002-0.005 M such as are used in the study of phospholipase action, it is even more likely that monomers are the chief, if not the exclusive, species of DHL in solution. ACKNOWLEDGMENTS The authors are indebted to Dr. Erich Baer of the Banting and Best Department of Medical Research, Toronto, whose generous gifts of the dihexanoyllecithin and dioctanoyllecithin made this study possible; and to Mr. Frank C. Wissler of the Biophysics Department for performing the ultracentrifuge runs. REFERESCES 1. BASGHAM, A. D., AA-D DAWSON, R. M. C., Biothem. J. 72, 486 (1959). 2. HANAHAN, D. J., AND JAYKO, M. E., J. Am. Chem. Sot. 74, 5070 (1952). 3. HaNAHAx, D. J., J. Biol. Chem. 195, 199 (1952). 4. HABERMANN, E.. Biochem. 2. 328, 474 (1957). 5. LONG, C., AXD PENNY, I. F., Biochem. J. 65, 382 (1957). 6. ROHOLT, 0. 4., AND SCHLAMOWITZ, M., Arch. Biochem. Biophys. 77, 510 (1958). 7. BAER, E., BUCHNEA, D., AND GR~F, T., Can. 1. Biochem. Physiol. 38, 853 (1960). 8. BAER, E., AND MAHADEVAN, V., J. Am. Chem. sot. 81, 2494 (1959). 9. GLICK, D., J. Bid. Chem. 156, 643 (1944). 10. BAER, E., AND KATES, M., J. Am. Chem. Sot. 72, 942 (1950). 11. RHODES, D. N., AIYD LEA, C. H., Biochem. J. 65, 526 (1957). 12. RHODES, D. N., .~XD LEA, C. H. in “Biochemical Problems of Lipids” (G. Popjak and E. LeBreton, rds.), p. 73. Interscience Publ., New York, 1956. 13. HANAHAN, D. J., RODBELL, M., AND TURNER, L. D., J. Biol. Chem. 206, 431 (1954).
ACTION
L.. ASD THOMAS, I. L., J. Chem. sot. 1958,483. MARPLES, E. A., AND THOMPSOX, R. H. S., Biochim. et Biophys. Acta 30,187 (1958). ~ANHETNI~GE~, u’. E., in “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. II, Part A, p. 355. Bcademic Press, New York, 1954. SUJINER, J. B., AXD SOMERS, G. F., “Chemistry and Methods of Enzymes,” p. 24. Academic Press, ?;em Tork, 1947. BJERHUX, J., SCHWARZENBACH, G., ASD SILLIN, L. G., Compilers, “Stability Constants of Metal-Ion Complexes,” Part I : “Organic Ligands,” p. 76. Special Publ. No. 6. The Chemical Society, London, 1959. HAWKE, J. C., Biochem. J. 64, 311 (1956). HUGHER, A., Biochem. J. 29, 437 (1935). DELEZENNE. C., AND FOURNEAU, E., BUM/. sot. c-him. 15, 421 (1914).
14. S~USDERS,
15. 16.
17.
18.
19. 20. 21.
OF PHOSPHOLIPASE
ON LECITHINS
379
22. MCBAIN, J. W., “Colloid Science,” pp. 244-251. D. C. Heath and Co., Boston, Mass., 1950. E., “Solu23. MCBAIN, M. E. L., .~ND HUTCHINSON, bilization,” pp. 28-43. Academic Press, New York, 1955. 24. JIRGENSOXS B., “Organic Colloids,” p. 60. Elsevier P;bl. Co., Amsterdam, 1958. 25. KLEVEIYS, H. B., J. Phys. & Colloid Chem. 52, 130 (1948). 26. EHRESBERG, A., Acta Chem. Stand. 11, 1257 (1957). 27. GOSTIX~. L. J.. .~XD MORRIS, M. S., J. Am. Chwn. Sot. 71, 1998 (1949). 28. GOSTINC, L. J., Advances in Protein Chem. 11, 429 (1956). 29. SAUNDERS. L., AXD THOMAS, I. L., J. Chem. sot. 1958, 483. in Bio30. SCHACHX~Y, H. K., “Ultracentrifugation chemistry.” Academic Press, New York, 1959.
OF
BIOCHEMISTRY
Studies
AND
BIOPHYSICS
364-379 (1961)
of the Use of Dihexanoyllecithin and Other as Substrates for Phospholipase A’
With Addendum
on Aspects of Micelle 0. A. ROHOLT
From
94,
the Division
AND
Properties
of Dihexanoyllecithin
M. SCHLAMOWITZ
of Research Biochemistry, Roswell Park Memorial for Cancer Research, Buffalo, New York Received
Lecithins
December
Institute
29, 1960
Some properties of the phospholipase A from venom of Crotalus durissvs terrificus have been studied using as substrate a water-soluble lecithin, dihexanoyl-L-a-glycerylphosphorylcholine (DHL). Ca++ is required for activity, and the concentration required for maximal activation is about 0.004 M at pH 6.8-9.8. The pH optimum for the system in the presence of excess Ca++ is pH 8. Ba++, Zn++ and Cd++ inhibited the reaction. Inhibition by Ba++ was competitive with Ca++, and ‘most probably involves a site on the enzyme. Caproate, one of the products of the enzymic reaction, has essentially no effect on the reaction even at levels 10 times that of the substrate. Hexanoyllysolecithin, the other product, had no effect when t,ested at 5 X 1Om6M. The influence of other lysolecithins , sodium dodecyl sulfate, Tween 20, and Tween 80 on the activity of the enzyme toward DHL, and toward the water-insoluble dioctanoyl-, dimyristoyl-, and ovolecithin is also described. Studies on the micellar properties of DHL, with estimates of t,he critical micelle concentration and micellar weight are also presented. INTRODUCTION
of Ca++ activator are restricted by the insoluble nature of the long-chain fatty acids and their calcium soaps, and by the micellar nature of the lysolecithins. Although the difficulties arising from the use of heterogeneous or impure lecithins can be surmounted by use of purified dipalmitoleyllecithin such as has been isolated from yeast (2), the complications associated with the water-insoluble nature of the high molecular weight lecithins remain. The finding of Hanahan (3) that natural lecithins are hydrolyzed in moist ether by phospholipase A has been of great value in studies of the enzyme and has provided a useful method for the preparation of lysolecithins. However, studies on the effects of ionic strength, pH, and of substances insoluble in ether are obviously not amenable to this medium ; nor is it known with certainty that all components of this system are in simple solution. In both the aqueous emulsion and ether systems enzymic hy-
St’udies of the action of phospholipase A on lecithin are often complicated by the nature of the substrate. Many lecithins from natural sources are heterogeneous with respect to their fatty acid residues, posing problems of substrate specificity. Further, most of the lecithins are insoluble in water and necessitate the use of multiphase systems in which the degree of dispersion of substrate and its relation to enzyme activity is poorly defined. Yet another element of undetermined significance for enzyme action on lecithin emulsions is the demonstration that micelles or emulsions of natural lecithins are able to ‘(adsorb” electrostatic charges (1). It may further be noted that with the lecithins of high molecular weight, studies of the influence of the hydrolysis products (fatty acids and lysolecithin) and ‘This investigation was supported in part by a research grant (No. C2585) from the National Institutes of Health, U. S. Fublic Health Service. 364
ACTIOS
OF PHOSPHOLIPASE
drolysis often does not proceed as a linear function of time, but manifests a lag period (4, 5) of undefined etiology. In an earlier communication (6)) dihexanoyllecithin (DHL) ,2 a short-chain lecithin was investigated for possible use as a water-soluble substrate for phospholipases. It was shown that DHL can serve as substrate for phospholipase ,4 and that neither a lag phase nor precipitation of the calcium soap is encountered. The present study further characterizes this system with respect to the pH for optimal activity and the influence of activator and inhibitor metal ions and of hydrolysis products. Some comparative studies are also presented of the action of phospholipase A on some of the water-insoluble lecithins, c.g., ovolecithin, dimyristoyllecithin, and dioctanoyllecithin. Observations of the influence of surfactants like Tween 20 and lysolecit,hins are also recorded. MATERIALS
PHOSPHOLIPASE A (PL-A) The (Miami
enzyme preparation was dried venom Serpentarium) from Crotalus tlurissus ter-
rificus.
LECITHINS Synthetic dihexnnoyllecithin (DHL), dihexanoyl-L-a-glycerylphosphorylcholine (8) was obtained from Dr. Erich Baer of the Banting Institute, Toronto. Anal. Calcd. for GH,?O&P: N, 2.97; I’, 6.57. Found N, 2.92; P, 6.62. Bnalysis for rholine (9) rhor;-ed 29.7% (as choline chloride) ; theory, 29.6%. Synthetic dioctanoyllecithin (DOL) was also obtained from Dr. Barr. D;myristovllerithin (DML), synthesized acrording to Baer ct r/l. (lo), Lamotte Chemical co. Anal. Calcd. for C,,H,,O,NP: N, 2.02; P, 4.45. Found N, 2.10; P, 4.41. ‘As may be noted by comparison with a previous paper (6), the nomenclature of the lecithins llas been changed. DHL was formerly designated as DCL. The change has been made to avoid confusion that, might arise from the use of the trivial names of the, fatty acid substituents of lecithins, in the G-C,, range. The present system conforms to the nomenclature in use (7) for lecithins and rcphnlins in this group.
OK LECITHINS
365
Ovolecithin (OvoL) was prepared by the chromatographic procedure of Rhodes and Lea (11) and stored in methanol-chloroform (30:70 v/v) solution at -10”. In one of several runs the elution peak was divided into three parts. The leading portion, the central portion, and the slowest moving portion were each analyzed for phosphorus, choline, and fatty arid. The expected 1:1:2 ratio for these constituents was found. The slowest moving portion is reported lo rontain the more saturated lec,ithins (12).
Myristoyllysolecithin (MLL) was prepared from DML by the action of the ycnom enzyme by a procedure based on the methods of Hanahan rt rtl. (13), and of Saunders and Thomas (14). A solution of venom in 0.005 ‘$1 CaCl, was incubated at room tempcraturc with a solution of DML in 4Yc ethanol in ether. The insoluble lgsolccithin produced was reprccipitated from chloroform with ether until crystalline, and finally was crystallized three times from hot ethanol. Ovolysolecithin (OvoLL) ~3s prepared from ovolecithin in a similar manner using the slower portions of the elution peak. The OroLL was recrystallized five times from hot ethanol. The preparation of the OvoLL involved the use of considerably more enzyme than did the preparation of the MLL; it was still contaminated with cnzyme ercn aftrr five recrystallizations (rf. Results, Fig. 6). Marplcs and Thompson (15) working with OroLL prepared using venom from the cottonmouth moccasin were able to eliminate adsorbed enzyme by recrystallization of the Iysolecithin.
CAPROIC ACID The caproic acid used was assayed by titration. Neutral equivalent: found 119; theory 116. METHODS
Assay OF PHOSPHOLIPASE A ACTIVITY Enzymic hydrolysis of the lf,cithins was followed by continuous titration of the liberated fatty acid at, constant pH, using a Beckman model G pH meter with extension probe electrodes. The assay system was standardized to 1 ml. total volume and contained lecithin, CaC12, ethylenediaminetetraacetic acid (EDTA), NaCI, buffer, and enzyme. The concentrations of the lecithins (0.002-0.005 JI) and of Ca” (O-0.02 M) will be specified for each experiment. It is important to note that these levels of substwtr arc in the range where vrry little if any dihexanoyllccithin is present as miccllrs (WC Atldcntlrc~n). EDTA (0.00071
366
ROHOLT
AND
SCHLAMOWITZ EXPERIMENTAL
AND
RESULTS
M) was used along with excess Cat+. The activity of the enzyme was increased several fold by its presence, presumably due to binding of inhibitory ions3 The NaCl (0.1 M) was used to minimize differences in ionic strength between experiments ; and the inclusion of a small amount of buffer (5 X 10m4 M maleate, Tris, or aminomethylpropanol) made it practicable to control the pH to within 0.2 pH unit during titrations. Enzyme solutions were prepared from the dried venom in 0.00355 M EDT$, dialyzed in the cold against several changes of 0.01 M CaCh, and then centrifuged clear. They contained about 5 pg. of dry venom/pi. The reactions and titrations were carried out under nitrogen in small vials with rapid mixing. A l-ml. Gilmont ultramicroburet calibrated to 0.0001 ml. was used for the titration with 0.100 N NaOH. Under these conditions there was no CO, blank; and preliminary experiments established that nonenzymic hydrolysis of substrate in the pH range used in these studies was negligible. It was further ascertained that formation of the titratable acid was not the result of the action of proteases on the protein of the venom. Titrations leveled off at values which corresponded approximately to the liberation of one mole fatty acid per mole lecithin and they could not be increased by further addition of 20-30 times the amount of venom normally used (15-30 pg.). In all the experiments in this study, complete titration curves were recorded relating the micromoles of NaOH taken up as a function of time of hydrolysis. In those experiments where the relevant effects are best shown by the complete curves (Figs. 5-7), they are drawn. In the others where the relations of interest are the relative activities (Figs. 1-3, Table I), the activities are expressed as the reciprocal of the “time values” (17), i.e., the reciprocal of the time (minutes) to achieve 20 or 30% hydrolysis of substrate, l/T, and l/T, Thus, in a reaction employing 1 ml. of 0.002 M substrate, a value l/T, of 0.2 represents the reciprocal of the time (5 min.) for 20% (0.4 pmole) hydrolysis. The l/T,0 and l/Tm values are on the linear or near linear portions of the hydrolysis curves. Reproducibility of titration data was satisfactory, generally within 5%, but occasionally varied by up to 15% (cf. Figs. l-4).
This relationship was established in the standard system with levels of venom solution ranging from 0.2 to 6.0 ~1. For the lower levels of venom solution (0.2-1.0 ~1.) the substrate concentration was 0.002 M DHL. For the higher ones the concentration of DHL was 0.005 M. In all cases the Ca++ concentration was 0.02 M and the pH was 8 (Tris buffer). The results, Fig. 3, show a linear relationship between activity and enzyme concentration over the entire range of enzyme concentration tested.
3 Large amounts of Zn++ has been reported in the native venom of several species of snakes (16). It is possible that the need for EDTA in these studies is related to this fact.
‘The Ca++ concentrations are corrected for the 0.71 pmole presumably bound by the EDTA present. The amount bound by the enzyme is considered to be negligible.
1. CHARACTERISTICS OF THE DIHEXANOYL-
LECITHIN-PHOSPHOLIPASE
Effect of Ca++
A SYSTEM
on the Activity
of PL-A
The enzyme from Crotalus durissus terrifkus venom was inactive in the standard system unless Ca+ +, in excess of the EDTA, was added. The influence of Ca++ was investigated at several pH levels with 5 ~1. enzyme solution and 0.005 M DHL. The buffers were maleate at pH 6.8, Tris at pH 7.8 and 8.8, and aminomethylpropanol at pH 9.8. The results, Fig. 1, show that over a wide pH range, maximal activity is achieved with 0.004 M Ca++ and that further increases in Ca++ up to 0.02 M are without further effect4 Optimal
pH for the Activity
of PL-A
A pa-activity curve was then obtained with the standard assay system containing 0.002 M DHL, 0.02 M Ca+ +, and 3 ~1. of venom solution. From pH 6 to 7, maleate buffer was used; Tris from pH 7 to 8.8, and from pH 9 to 10 no buffer was necessary. The curve, Fig. 2, is symmetrical with a broad maximal region centering around pH 8. Relation
between Enzyme Concentration and Rate of Hydrolysis
ACTIOh’
OF PHOSPHOLIPASE
367
ON LECITHIKS
pH 9.9
FIG. 1. Activation
Temperature
of phospholipase
Coeficient
A by Ca++ at several
pH values. See text for details.
of Activity
The standard system was run at 20, 30, and 40” at pH 8.0 (Tris buffer) with 0.005 Ilf DHL, 0.005 ,I[ Ca+ +, and 3 ~1. venom. The temperature coefficient of the activity ( l/Tso) per 10” interval was 1.55 and 1.45 for the 20-30” and 30-40” intervals, respectively, and the energy of activation calculated from the Arrhenius equation was 7300 cal./mole for the hydrolysis of DHL by this enzyme. Influence of Some Metnl Ions on the Activity of PI,-A The effect of Mg++, Ba++, Zn++, Cd++, &In++, Co++, and Cu++ was tested at several concentrations and pH values in the standard system with 0.005 M DHL and 0.005 M Caf f. This level of Ca+ + (only slightly in excess of the minimum re-
FIG. 2. Activity of phospholipase noyllecithin as a function of pH.
-4 on dihexa-
quired to give maximal activation, cf. Fig. 1) was selected to permit effects of the other cations to be manifest. None of the ions tested increased the activity beyond the level already achieved by the Ca+ -1.present. At pH 8, Mg++ (0.005,
368
ROHOLT
ENZYME
AND
SCHL.4MOWITZ
CONCENTRATION
( x venom solution)
FIG. 3. Rate of hydrolysis of dihexanoyllecithin as a function of enzyme concentration. Activity values on the two plots are not comparable since the substrate concentrations are different. See text for detail:
0.02, and 0.05 M) did not affect the reaction rate. Mn++ (0.005 M) did not inhibit but gave a small precipitate even when the pH was lowered to 6.8. Co++ (0.001 and 0.005 M) and Cu++ (0.005 M) were inhibitory (50-90%) at pH 6.8 but were not amenable for further study because they too gave precipitates at this pH. Ba++ at pH 8 was found to inhibit the reaction without forming any precipitates and was thus suitable for a study of the kinetics and mechanism of its inhibitory action. If the inhibition is the result of a competitive relation between Ba++ and Ca++, it will be determined by the (Ba++) : (Cat-+) ratio rather than by the absolute concentrations of the ions. Activity at pH 8 was measured at 0.005 and 0.002 M Ca+ + for several concentrations of Ba++. The concentration of DHL was 0.005 M, and 3 ~1. of venom solution was used. A plot of activity versus (Ba++) : (Ca++) ratio, Fig. 4 curve (a), shows that the degree of inhibition depends on the (Ba++) : (Ca++) ratio and is independent’ of the individual ion concentrations. Verification of this competitive relationship is also seen in the straight line obtained in the plot l/w versus (Ba++): (Ca++) in Fig. 4 curve (b). *4 study of the inhibitory action of Zn++
at pH 8 was ruled out by the formation of a heavy precipitate. At pH 6.8, however, no precipitate formed. At this pH the reaction could be inhibited 95% by 0.00085 M Zn+ +5 and released by addition of sufficient EDTA to bind the Zn+ +. In this experiment, Fig. 5 curve (a), 5 ~1. of venom solution was added to 0.72 ml. of a solution containing 5 pmoles DHL, 0.71 rmole EDTA, 5 pmoles Ca+ +, NaCl, maleate buffer, and 0.85 pmole Zn+ +. Under these conditions the reaction was inhibited by the Zn++. After 8 min. 0.27 ml. of a solution containing 0.6 pmole Ca++ and 0.71 pmole EDTAG adjusted to pH 6.8, was added. The total amount of EDTA present (1.42 pmoles) exceeded the amount of Zn++ (0.85 pmole) and, because of its great avidity for the metal, reduced the level of free Zn++ to a noninhibitory level. The enzymic reaction then proceeded at about the control rate [compare with curve (b)]. A second quantity of Zn++ (0.85 pmole) in a volume of 0.017 ml. was added 5 min. later. Again the total amount of Zn++ (1.7 pmoles) slightly exceeded the total amount of EDTA (1.42 pmoles) , and again the enzymic reaction essentially stopped. After 3 min., a second portion of Ca-EDTA solution was added, and as before the enzymic hydrolysis resumed at about the same rate as the control.
‘The stability constants for Zn-EDTA and Ca-EDTA are approximately 10” and lOlo, respectively (18). Since the reaction mixture contains 0.00071 M EDTA which binds the Zn++ tightly, the actual concentration of Zn++ is closer to 0.0001 M.
‘EDTA was added as the Ca chelate (pH 6.8) to avoid the liberation of H+ (0.7 wmole) which would have resulted if the NasH (EDTA) salt were used to chelate the Zn++. The additional trace amount of Ca++ contributes little to the already maximally stimulating concentration present.
ACTION
OF PHOSPHOLIPASE
ON LECITHIM
369
- 25
1 5
FIG. 4. Relation between t,he ratio of [Ba”] : [Ca-+] and the activity of phospholipaae Curve (a), activity versus [Ba++]:[Ca++]; curve (b), l/v versus [Ba‘*]:[Ca++]. O-0, o-e, 0.005 M ca+-; U---O, B-I, 0 02 31 Cat+. See text for detail.
TIME
A.
(minutes)
FIG. 5. Effect of alternate additions of inhibitor ions (Zn++ or Cd-‘) and of EDTA on the rate of hydrolysis of dihexanoyllecithin by phospholipase B in the presence of Cat+. Curve (a), inhibitor ion, Zn”; curve (b), control for curve (a). Curve (c), inhibitor ion, Cd++; curve (d) control for curve (cl. See text for details on amounts and order of additions of inhibitor ions and EDT$.
Similar experiments were carried out at pH 8 with Cd+ ii using 3 pl. venom. Though ‘The sentially
stability constant for Cd-EDTA the same as for Zn-EDTA (18)
is es-
somewhat less effective than the inhibition produced at pH 6.8 by Zn++, inhibition by Cd+ + at pH 8, Fig. 5, curves (c) and (dj , could also be counteracted by EDTA.
370
ROHOLT
AND
SCHLAMOWITZ
TIME
(minutes)
2.ot-
1.8 0.0 1.6 0 I .4
0 n .-----
M OVOLL.
5Xlo+M
OVOLL.
3.3Xl0‘4M
OVOLL.
3xto-3rd OVOLL. 8XIO-3M OVOLL. 5X10- M MLL.
1.2 i 2
1.0
i 32 0.8 E 25
0.6
FIG. 6. Comparisons of the enzymic hydrolysis ovolvsolecithin and in its absence (0.0 M OvoLL). is also shown.
Influence of Caproate on the Activity of PL-A The effect of caproate, a product of the action of PL-A on dihexanoyllecithin was determined in the standard assay system with 3 ~1. of venom solution, 0.005 M DHL, and 0.02 M Ca++ at pH 8 (Tris buffer). At concentrations of 0.001, 0.01, and 0.05 M corresponding to 0.2, 2, and 10 times the initial DHL concentration, caproate caused less than 10% inhibition of the reaction rate. In,fluence of Lysolecithins on the Hydrolysis of Dihexanoyllecithin by PL-A Figure 6 depicts the hydrolysis of DHL in the standard assay system containing 0.002 M DHL, 0.02 M Ca++, and 3 ~1. venom solution at pH 8 (Tris buffer) in the presence of ovolysolecithin (1 x 10e5 to
of dihexanoyllecithin in the presence of The effect of myristoyllysolecithin (MLL)
8 x low3 M)s and in its absence (0.0 J!f OvoLL). The Ca+f was added 10 min. prior to the addition of the enzyme. It is seen that compared with hydrolysis in absence of OvoLL, hydrolysis of DHL is increased roughly 3-7-fold by the presence of OvoLL. The significant ‘Lblank” during the preincuba,tion period, due to residual enzyme activity in the OvoLL (see Materids), seen at the two higher levels of OvoLL (0.008 and 0.003 M) becomes negligible at lower levels of OvoLL. The possibility that the increased hydrolysis of DHL by the presence of OvoLL might be due to the presence of some naturally occurring stimulatory substance in the OvoLL was ruled out by a comparison of *Solutions of lysolecithins were used within 24 hr. since it was observed with both MLL and OvoLL that precipitates formed after 2 or 3 days at room temperature.
ACTION
OF PHOSPHOLIPASE
these results with the ones obtained with myristoyllysolecithin enzymically prepared in ether from synthetic dimyristoyllecithin (Uoterials). At 5 x 10-j M it is seen that MLL and OvoLL produced comparable effects (Fig. 6). To further insure that this property of both OvoLL and MLL was not in any way related to their preparation in the ether system, a sample of MLL was prepared by the action of the enzyme on DML in an aqueous system and tested as follows: a suspension containing 10 pmoles DML in 0.01 iM Ca++ at pH 8 (Tris) and 4 ~1. venom solution was incubated according to the standard assay procedure for a period of 160 min. (94% hydrolysis). Portions of this reaction mixture (0.06,0.006, and 0.002 ml.) sufficient to produce final concentrations of 5 x 10-4, 5 x 10-j, and 1.7 x lo-” M MLL in the standard procedure were t,hen tested using 0.002 M DHL substrate, 0.02 M Ca++, and 3~1. venom solution at pH 7.8 (Tris buffer). The MLL prepared in this manner stimulated the reaction the same as did the MLL and OvoLL prepared by the ether system. Hexanoyllysolecithin, the normal product of reaction of PL-A on DHL, prepared in water as was the MLL, had no effect at a level of 5 X 10Ws M. It is seen from these studies that lysolecithins derived from synthetic DML and from OvoLL greatly increase the activity of Crotalus durissus terrificus venom PL-A toward DHL. A similar effect of OvoLL on the activity of purified bee venom PL-A toward OvoLL was attributed by Habermann (4) to finer emulsification of the substratc. It, is unlikely that such an interpretation applies in the present study since the dispersion of DHL at the concentration used here is essentially complete (see Adden&m). A satisfactory explanation for the above observations is not readily apparent. Influence of Sodium Dodecyl Sulfate (SDS), Tween 80 and Tween 20 on the Hydrolysis of Dihexnnoyllecithin by PI,-A Inasmuch as lysolecithins are surfacc-actire agents in addition to having structures related to the substrates for PL-A, ot’her
ON LECITHINS
371
surface-active agents were investigated for possible stimulatory action. The substances were test,ed in the standard assay procedure using 0.002 M DHL and 0.02 M Ca++ at pH 8 (Tris buffer). The conditions used and the results are given in Table I. In the presence of SDS, (0.0026 or 0.037 111) hydrolysis was completely inhibited when as much as 500 ~1. venom solution were used. Tween 80 at 0.26% and Tween 20 at 0.038 and 0.75%, had little effect on the hydrolysis. At 3.8%, Tween 20 inhibited the reaction about 46%. This inhibition was not altered or reversed by the presence of 5 x 109” J!1 JILL. Action of PL-A on Dihexanoyllecithin 4% Ethanol-Ether Solution
in
This experiment was carried out to establish whether DHL could be hydrolyzed by PL-A in the ether system the same as were the higher molecular weight lecithins. Venom (10, 50, and 100 pg. in 0.01 ml. of 0.01 M CaC12) was added to samples (1 ml.) of the ethanol-ether subst’rate solution (2 ~molcs DHL) , and the reaction was followed at, room temperature by titrations of the mixture according to Hanahan et al. (13). The reactions proceeded without any lag period. Relat,ive initial rates of 1.0, 0.5, and 0.03, respectively, were observed for 100, 50, and 10 pg. venom, and hydrolysis proceeded to about 8076 of theoret’ical. 2. COMPARATIVE EFFECTS OF MYRISTOTLLYSOLECITHIN.~ND OTHER SVRFACE-ACTIVE COMPOUA-DSONTHE A4c~rvr~r OF PHOSPHOLIPASW,A TOWBRD DIOCT~~NOYL-, DIM~RTSTOYL- U-D OVOLECITHIN
The following experiments describe t,he influence of myristoyllysolecithin (MLL) , sodium dodecyl sulfate, and Tween 20 on the action of PL-A on some of the waterinsoluble lecithins. Influence of MLL on the Hydrolysis T)OL, DML and Oz~oL by PL-A
of
The effects of MLL mere tested by the standard assay procedure with 0.002 Jll of the appropriate lecithin and 0.02 ,W Ca++ at pH 8 (Tris buffer). Several concent,rations of JILL and of v(lnom were used, and
l)Ol. Dhll, OVOL
Twain
20 3.8’ ;, :1.cv:; 3.8“(
0.03b 3” 3
0.x r 0.0
0.05 c 0.22
85 _
* For the control w-here the rate \VAS too fast to measure, the value 27 was ot)t:Cnecl hy cdculation. 500/S x 0.16. for 5 or 90 niin. or 20 hr. at 5” did not b I’sposure of venom to 3.8’,&* Twecn 20 :tt room 1emper:iturt:s wlwn assayed against I)IIl.. There :we :tppnrent Iy no irrrversihle significantly change the activity chnnges of the enzyme in 3.8’,‘, Twcn 20. c SIX I:ig. 7 for comparison of control and cspcrimcntnl rates.
the rcwlts obtnintd are suuuuarizcd in Table II together with thaw previously obtained for hydroly& of L)HL. Scvcrsl p0int.s niny bc noted which focus attention on the colliplcxity of thrx various systmis. i\ltt~ougti Yolubility of I)Hl, is aplxmntly not n factor, its rntc of hydrolysis is incrcawl scvcml fold in ttw t)rcwirc of NLL. On tlw ottwr hnntt, ciuulsiona of INI, arc cou~tktcly clnrific>tt by 2.3 X IO-: -11 JILL. but its ratr of hydrolysis is not, nffcctcd. \Yith I>JIL as it substratc~. tticro is no visiI)lc clffwt on the solubility at the lower conwntrations of 11I.T., but ttw lag twiod in ttw tly(lrotysis is rcduwd. Xt ttw highwt concwitrntion of JII,I. the D.\IT, is ahno& couipl~~tcl~ solubilizcd and the tag is climiriatcd. In the cab0 of 0~01,. cwlntially no hydrolysis owurs in tlic abetmcc of MI,I,. In its t)rcwncc~ hydrolysis ia incoulptctcF but tmth thcl r:lk and the mtmt of hydrolysis arc’ incrcawd with incrwsing amounts of JILL, and no lag period i:: seen. .L\t. 5.6 X IO-” M 1lI,I,, hydrolysis of 0~01, falls off txpidt\- :iftcr BOY wuiptrtion. but can bc
drivcu to c~ou~plction if 85 pl. \-morn solution txtlw than 3 pl. is used. The Z,~jlrmce of Tuwn
“0 on the llydroly-
TIIO level of Twecn 20 (3.8%) which C:LIIS:(Y~ about. 469;; inhibition of hydrolysis of DHL has bwn reported to almost wint)lctcly clarify suspensions of ovolccit.tiin (,4j. A comparkon of its cffccts on souio of tile othrr lccittiins was invcstigatcd. Town 20 (3.8“; ) t)rougtit about almost complete clarification of emulsions of I)OL (0.002 JZj~ DMI,, and 0~01~. The conq~lcx nature of thaw systmis tiowcvor is again Aown by the: variety of c1ffect.sseen for the reaction rat.es. With 1101, the reaction rate war; inhibited 83% (Table I). \\Yth DJlI,. that tlydrolysk rate was virtually unchanged Imt tlw lag pcriotl was eliminated I Fig. 7j. \\‘ith OvoI,, ttic hydrolysis rntc was iricwnsrd from the zero lev(ll of the control t,o n value c*omparablc to that given by hytlrolyk of DHI, (,Tnblo I 1.
ACTION
OF PHOSPHOLIPBSE
ON LECITHINS
373
TABLE II INFLUENCE OF MYRISTOYLLYSOLECITHIS ON THE HYDROLYSIS OF DIHEXANOYLLECITHIN, DIOCTANOYLLECITHIN, DIMYRISTOYLLECITHIN AND OVOLECITHIN BY PHOSPHOLIPASE A Substrate
Venom solution
0.002Y
A4LL
d.
M
DHL DOL*
3 0.03
5 x 10--b 2.3 x 10-a
DMLc
0.03
3 x 10-E
DML
0.03
DML
3
5 x 10-j
DMI,
3
1.7 x 10-a
OvoLe OVOL OVOL
3 3 3
5 x 10-b 8 x 10-d 5.6 x 10-s
OVOL
85
5.6 x 10-S
10 x 10-j
Comments” Increase in rate (see Fig. 6). Emulsion solubilized, but neither rate nor extent of hydrolysis influenced. 15.min. lag, slow reaction, hydrolysis essentially stops at about 50Gj, of completion.d 5-min. lag, slow reaction, hydrolysis essentially stops at about 50y0 of complet,ion.d Lag period short,ened, but no effect on subsequent rate or solubility. Lag period eliminated but no effect on subsequent rate; reaction mixture opalescent instead of unstable emulsion. Same as control; almost no action. Reaction rapid up to 25% hydrolysis, then stops. Solution clear, reaction rapid to GO?&hydrolysis, then very slow. Solution clear, 90% hydrolysis in 5 min., complete in 10 min.
a Compared to controls, in absence of myristoyllysolecithin. * In controls, DOL forms an emulsion when shaken in water, but separates readily into two layers. It is nevertheless attacked smoothly and about 70 times more rapidly than is DHL. The rate remains nearly constant until about 75% hydrolysis is achieved, after which the rate falls off slowly. At the completion of hydrolysis, the emulsion has disappeared and the reaction mixture is clear. c DML is not soluble but forms a suspension. Hydrolysis is preceded by a lag period. For a typical control curve, see Fig. 7. The rather abrupt increase in rate is presumably associated with the formation of some critical level of MLL, a product of the reaction, since, as seen above, the addition of 1.7 X 10m3 M MLL eliminates this lag period. d With 0.03 ~1. venom solution no hydrolysis is observed over a 30.min. period in the absence of MLL. 8 The ovolecithin forms a coarse suspension. The control rate is very slow; only about 57, (0.1 rmole) is split in 30 min. Although OvoLL, a product of its hydrolysis, activates the DHL system (Fig. 6), there does not seem to be any effect on the hydrolysis of OvoL by the OvoLL formed during the reaction here. DISCUSSION
Most lecithins isolated by lipid extraction procedures from natural sources are insoluble in water and contain saturated and unsaturated fatty acid residues of 16 or more carbon atoms, although phospholipids of bovine muscle and brain have been reported to contain small amounts of volatile fatty acid residues (19). It is thus conceivable that water-soluble synthetic lecithins like dihexanoyllecithin are model substrates for phospholipase-A in only a limited sense. But it is equally conceivable that even with the higher molecular weight lecithins, only that minute amount soluble in water and in equilibrium with t’he insoluble phase serves
as a substrate. In any event, the practical difficulties attending the use of the waterinsoluble lecithins (see I~~troductian) recommends the use and more extensive inwater-soluble vestigation of simplified model compounds like dihexanoyllecithin for studies of the mechanism and kinetics of lecithinasc action. It is seen, for example, from the hydrolysis curves of DHL and DML (Figs. 6 and 7)) that hydrolysis of the former proceeds without the complication of a lag period. As mentioned earlier, lag periods and failure to demonstrate linear relationships bctween enzyme concentration and rates of hydrolysis have been observed’> with high
ROHOLT
TIME
AND SCHLAMOWITZ
(minutes)
FIG. 7. Effect of Tween 20 (3.8%) on the hydrolysis of dimyristoyllecithin by phospholipase A (3 ~1. venom solution). O----O, control; X.---X, Tween 20. The curve obtained in the presence of Tween 20 has been displaced 8% min. to the right (Iower abscissa) to facilitate comparison of the steep portions of the two curves.
molecular weight natural lecithins as well (4,5). The pa-activity curve for DHL is symmetrical; and the optimum pH value, pH 8, would appear to be governed by the ionieation of as yet unidentified groups in the enzyme since the ionization of both the phosphate and choline groups of the substrate do not change appreciably in this pH range. An optimal value of pH 8.3 has been reported (4) for the enzyme from bee venom acting upon egg yolk. It was not established for that system whether the marked asymmet,ry of the curve and rapid fall in activity on t,he alkaline side of the optimum pH value was a cha,racteristic of the particular enzyme, or due to the state of emulsion of substrate as a function of pH, or to a reduction of free Ca*+ with increasing pH. The optimal pH for the activity of the phospholipase A from black tiger (Note&s
scu,tatus) venom is reported by Hughes (20) t’o be pH 7.8. In regard to the known (5, 21) requirement of Ca++ for activation of phospholipase A, the inhibition by Ba++ indicat’es that activation by Ca++ involves a site for which the two ions are able to compete (Fig. 4). This competitive relation may be satisfied by either of two mechanisms. In the first case the action of Ba++ is viewed as competing with Cat+ for the formation of an active Cat+-enzyme complex which catalyzes hydrolysis of the substrate. The second mechanism postulates the formation of a substrate-enzyme complex which is activated by Caf + but not by Ba++. For the first case E + Ca++ @ ECa (active); (E) (Ca++)/(ECa)
= K,
(1)
ACTION
ECa + S *
OF PHOSPHOLIPASE
ECaS + ECa + P; (ECa) (S)/(ECaS)
E + Ba++ +
= K,
(2)
EBa (inactive);
(E) (Ba++)/(EBa)
= Kb
(:3)
and the equation for conservation of enzyme is E tc,tnl= Efree + ECa + ECaS + EBa.
(4)
Efrer is essentially zero since the activity is the same at 0.005 and 0.02 M Caf+ (Fig. 1). If the remaining forms of the enzyme arc expressed in terms of ECaS, the active one, and substituted into Eq. (4), one obtams E total o=(6)+
KS
(Ba++)K,K, 1 + (Ca++)Kb(S)
(5)
Substitution of Vrn for hEtotal and II for k (ECaS) , in Eq. (5 ) and rearrangement of terms gives 1 11
(Ba++)K,K, (Ca++)Kb(S)V,
as the kinetic expression for the first mechanism. In an analogous manner, the second mechanism leads to 1 -= V
(Ba++)K,. (Ca++)KbV,
’
1 fi
(7)
A derivation based on both cases operating simultaneously also leads to Eq. (7). According to either Eq. (6) or (7)) the reaction is competitive for an “active” form of the enzyme, and a plot of l/v versus (Ba ++)/(Ca+ +), should give a straight line.g This was found to be the case [Fig. 4, curve (b)]. The data tend to rule out the likelihood that the actual substrate is a Ca-DHL complex and that Ca++ and Ba++ compete for the DHL. Under these conditions free Ca++, Ba++, and DHL would no longer be independently variable, and a plot of l/v versus (Ba++)/(Ca++j would not be linear. ’ The stoichiometric concentrations are used inasmuch as only small amount~s of the total Ba” and Ca’+ could he bound hy the small amounts of enzyme used.
OS LECITHINS
375
The very low levels of Zn++ or Cd++ capable of producing inhibition also point to primary involvement of a site on the enzyme rather than the substrate. If the relative binding of these two ions and of Ca++ by the enzyme differs by a factor of about lo6 such as is the case for their relative binding by EDTA, it would be clear why low levels of Zn++ and Cd++ inhibit the enzyme even in the presence of 50-fold levels of Ca+ + and why a demonstration of competitive inhibition at this st,age of purity of enzyme would be difficult. Such studies must await further purification of the enzymes. Yet another mechanism for apparent Ca++ activat,ion was considered by Long and Penny (5), i.e., that the fatty acids liberated by hydrolysis of lecithins are inhibitory and that Ca++ acts by combining with them. They showed such a role for Ca++ to be unlikely at least in the ether system inasmuch as maximal activation was achieved with Ca++ to substrate molar ratios of less than 1: 10, and, further, the fatty acids remained in the ether supernatant whereas the Ca+ + was carried down with the lysolecithin. This mechanism is equally improbable for the aqueous system using dihexanoyllecithin for several reasons. First, caproate, the fat,ty acid liberated by hydrolysis, exerted no inhibitory influence even at levels 10 times that of the substrate. Secondly, such a role for Ca++ could have been fulfilled by several of the other ions tested ; yet,, this was not’ the case. They were either inert or inhibitory, even in the presence of othcrwiee adequate levels of C:b+ +. Tho pattern of effects for the action of lysolecithins, sodium dodecyl sulfate, and Tween 20 on dihexanoyllccithin and on some of the insoluble lecithins is complex, and a clarification may have to await elucidation of the mechanism of action of the phospholipases and of t,he micellar properties of some of these surfactants and other lecithins. ADDENDUM
ASPECTS OF MICELLE PROPERTIES OF DIHEXANOYLLECITHIN Although consideration
DHL gave clear solutions in water, of its lipid structure raised the ques-
376
ROHOLT
ilxD
SCHLAMOWITZ
DHL (moles per liter)
FIG. 8. Relation
of DHL
concentration
to difference
in refractive
index between
solvent
and solution. tion of whether it might be present in micelle form. This point is relevant to the study on the action of phospholipase since the use of a substrate under conditions where part or all of it exists as micelles introduces new variables, e.g., the number and concentration of substrate species, and their susceptibility to enzyme action. Studies of refractive index increments versus concentration of solute, and of the diffusion patterns of DHL, both indicated that micelle formation occurs, but that under the conditions used the critical micelle concentration range, CMC,‘” is at about 0.011 M DHL. The concentrations used for the phospholipase experiments were 0.002-0.005 &f.
Relation
of Concentration of DHL to Refractive Index
For micelle-forming systems a change in the refractive index increment is observed at the CMC when this parameter is plotted as a function of solute concentration (25). The relative refractive index of solutions of DHL in an aqueous solution, pH 8, containing 0.02 M CaCl,, 0.01 J4 KaCl, 0.0007 M EDTA, 0.0005 J4 Tris, and 0.0125 &I NaOH was measured at 25” in a Brice-Phoenix differential refractometer, model BP-1000-V, at 436 mp. The instrument is sensitive to refractive index differences, in, of approximately 3 X 10.‘.
The data relating DHL concentration to An, shown in Fig. 8 was fitted best by two straight lines. Below 0.012 M the method of least squares gave a line passing through the origin with a slope of 0.0637. Above 0.012 kf a line with slope 0.0605 and intercept 38 X 10.” was obtained. The intersection of these two lines at 0.011 M represents the CMC range (25). The difference of about 5% in the slopes of the lines is similar to the values of 3.2-7.6% calculated from data of Klevens [Table I in Ref. (25)] for other micelle systems.
Diffusion
Studies with DHL
Diffusion experiments on solutions of DHL at concentrations above and below 0.011 M provided additional information on micelle formation and micelle-monomer relationships. These were carried out using a synthetic boundary cell in the Spinco model E ultracentrifuge at 19.5-21” with a 0.2-0.8” lo The narrow range of solute concentration in which micelle formation begins has been designated as the critical micelle concentration (CMC). Above this concentration both micelles and monomers exist and the dependence of many properties, e.g., osmotic pressure, conductivity, refractive index (22-24), on solute concentration is different from that observed below the CMC.
ACTION
OF PHOSPHOLIPASE
range in any given experiment, and photographs were taken at 4-min. intervals for 72 minutes. Figure 9, (a) and (b), shows representative patterns obtained when solutions of DHL at conc.entrations (0.0233 and 0.0339 ill) exceeding the CMC range indicated by the previous experiment were allowed to diffuse into the salt-buffer solvent. In both cases, a slowly spreading peak with a more rapidly spreading shoulder on the solvent aide of the peak was observed. It was found from the ratio of the area under the shoulder to the total area (obtained by drawing a vertical line at the shoulder-peak junction on all patterns) and from the t,otal conccntration of DHL, that the material represented by the shoulder at both concenirntions was equiralent to 0.011 A4 DHL (0.0103-0.0113 A4 for 0.0233 121 DHL; 0.0104-0.0120 M for 0.0339 A!1 DHL).‘l This corresponds to the value of the CMC range obtained independently from the refractjive index study. The constancy of this value at both concentrations of DHL is incompatible with the diffllsion behavior for a mixture of independent species. Although a theoretical treatment of diffusion for nonindependent species is not available for rigorous testing, the shoulder effect and the independence of the estimated CMC on concentration suggests the operat,ion of a system in which tlrrre is a rapid equilibrium bet\veen “monomerP and micelles, and in which micelles form above :I given concentration range, the CMC. The concentration gradient of the more rapidly diffusing “monomer” species is represented by the shoulder and, at levels greater than the equilibrium conrentration, the rnicelle gradient is shown by the peak. The vertical line referred to above marks this “monomerJ’-micelle equilibriunl concentration within the over-all DHL gradient in the spreading boundary. Based on this interpretation and the quantitative ~.aluc of the CMC obtained by the two methods, it was anticipated and found that diffusion of DHL at a concentration below the CMC range (0.011 .LI) yicldcd only a single rapidly spreading symmetrical “monomer” peak, Fig. 9(c) ; and that when a boundary was established between two different DHL concentrations, both greater than the CMC so that the “monomer” concentration would bc the same on both sides, only a micelle concentration gradient giving rise to a single slowly spreading symmetrical peak was seen, Fig. 9(d). ‘I No correction was made for the change of &/AC at, the CMC. This change is only about 5%. ‘* “Monomer” will be used to designate (DHL),, where for the predominant spcdies, n equals 1.
ON LECITHIKS
FIG. 9. Diffusion
patterns
of DHL.
(n) and (b), DHL at concentrations grcatel than thr CMC; 40 min. after boundary formation ; 20,410 r.p.m.; phase Illate angle, 55”. (c), DHL at a c~on~entration less than the CMC; 44 min.; 16,200 r.p.m.; phase plate angle, 40”. (d). diffusion across boundary formed by two solutions of DHL, both at concentrations grratol than !hc CMC; 44 min.: 20,410 r.p.m.; phase plate ang!c, 55”. Srfs text for intcrprclation. Diffusion coefflcicnts for the “monomers” and micselles were calculated from the data represented by Figs. 9, (c) and (d). For the micelles. values of 1.05 X IO-” and 1.06 X 10.’ s:,. cm./sec. were obtained by the “maximum ordinal e-arca” and methods (26), respectively. “maximum ordinate” For the “monomers” the> values from the average for three runs were 3.67 5 0.09 X 10.” and 3.66 -C 0.17 X IO-” scl. cni./se?.
Control
Diffusion Studies with Sucrose and Potnssiurn Law-ate
The lirnited the use of the
availability of DHL neressitated ultracentrifuge for the diffusion
378
ROHOLT
AND
studies though other systems not ‘involving centrifugal fields would have been preferred. To test the essential reliability of the results and to rule out technical artifacts, e.g., convection during synthetic boundary formation and boundary sharpening during runs even at the low speeds used, two control substances were investigated : sucrose, a simple substance with well defined diffusion characteristics; and potassium laurate, a micellar substance of defined CMC (25) not too different from the CMC of DHL. A single symmetrical peak was observed when a 0.78% aqueous solution of sucrose was allowed to diffuse into water for 44 min. Values of 5.28 X 10-O and 5.21 X 10.” sq. cm./sec. were obtained for the diffusion coefficient by the “maximum ordinate-area” and “maximum ordinate” methods, corrected to 25”. This is in good agreement with 5.20 X 10-O sq. cm./sec., the value for a 0.75% solution of sucrose at 24.95” obtained by extrapolation of the data of Costing and Morris (27). With potassium laurate (0.0555 &l diffusing into water) a shoulder was seen on the solvent side of the boundary such as was seen with DHL [cf. Fig. 9(a) and (b)]. The area of the shoulder comprised 45 f 1% of the total, equivalent to a potassium laurate concentration of 0.025 M. A similar pattern and a value of 0.025 M for the shoulder were obtained when the diffusion of a 0.048 M solution was carried out in the elect,rophoresis cell at 26”. This value of 0.025 M for the CMC range of potassium laurate obtained by the area analysis under both these conditions agrees with the reported CMC, 0.0255 M obtained by Klevens (25) by the refractive index versus concentration method, in which convection, formation of boundaries, and diffusion itself do not enter.
Sedimentation
Studies with DHL
A sedimentation run of 40 min. duration, carried out in the synthetic boundary cell at 59,780 r.p.m. on DHL at 0.005 M, a concentration below the CMC, showed only a rapid spreading of the boundary but no sedimentation. On the other hand, when a micelle concentration gradient was established by overlaying 0.0339 M DHL with 0.0170 M DHL (see Difksion Studies) sedimentation was demonstrated at 59,780 r.p.m., and a sedimentation coefficient of 0.83 S was calculated. Estimates of the maximal molecular weight for the micelle and “monomer” species of DHL may be obtained from the ralucs of the diffusion coefficients (281, assuming as a first approximation the Stokes-Einstein equation for rigid spherical particles, D = RT/GqrN, and the relation betpeen the radius, r, of the sphere and its molecular weight, M; T = (3Mp/4rN)1’S. The viscosity of
SCHLAMOWITZ the medium was taken as 0.010 poise, the value for water at 20”, and the values 6.02 X lo”, 8.3 X 10’ ergs/deg. and 293” Absolute, were used for N, R, and I’ respectively. For 8, the partial specific volume, a value of 0.91 ml/g. was used, assuming the density of solid DHL to be the same as OvoLL, 1.095 g/ml. (29). From these data a “monomer” weight of 560 and micellar weight of 24,000 were calculated. The micellar weight calculated from the sedimentation and diffusion coefficients (30) was found to be 21,400. These data indicate that there are about 45-50 DHL molecules per micelle. Considering that DHL may not be either rigid or spherical, the value of 560 for the “monomers” is in fair agreement with the formula weight of DHL, 472. At concentrations even further removed from the CMC, 0.002-0.005 M such as are used in the study of phospholipase action, it is even more likely that monomers are the chief, if not the exclusive, species of DHL in solution. ACKNOWLEDGMENTS The authors are indebted to Dr. Erich Baer of the Banting and Best Department of Medical Research, Toronto, whose generous gifts of the dihexanoyllecithin and dioctanoyllecithin made this study possible; and to Mr. Frank C. Wissler of the Biophysics Department for performing the ultracentrifuge runs. REFERESCES 1. BASGHAM, A. D., AA-D DAWSON, R. M. C., Biothem. J. 72, 486 (1959). 2. HANAHAN, D. J., AND JAYKO, M. E., J. Am. Chem. Sot. 74, 5070 (1952). 3. HaNAHAx, D. J., J. Biol. Chem. 195, 199 (1952). 4. HABERMANN, E.. Biochem. 2. 328, 474 (1957). 5. LONG, C., AXD PENNY, I. F., Biochem. J. 65, 382 (1957). 6. ROHOLT, 0. 4., AND SCHLAMOWITZ, M., Arch. Biochem. Biophys. 77, 510 (1958). 7. BAER, E., BUCHNEA, D., AND GR~F, T., Can. 1. Biochem. Physiol. 38, 853 (1960). 8. BAER, E., AND MAHADEVAN, V., J. Am. Chem. sot. 81, 2494 (1959). 9. GLICK, D., J. Bid. Chem. 156, 643 (1944). 10. BAER, E., AND KATES, M., J. Am. Chem. Sot. 72, 942 (1950). 11. RHODES, D. N., AIYD LEA, C. H., Biochem. J. 65, 526 (1957). 12. RHODES, D. N., .~XD LEA, C. H. in “Biochemical Problems of Lipids” (G. Popjak and E. LeBreton, rds.), p. 73. Interscience Publ., New York, 1956. 13. HANAHAN, D. J., RODBELL, M., AND TURNER, L. D., J. Biol. Chem. 206, 431 (1954).
ACTION
L.. ASD THOMAS, I. L., J. Chem. sot. 1958,483. MARPLES, E. A., AND THOMPSOX, R. H. S., Biochim. et Biophys. Acta 30,187 (1958). ~ANHETNI~GE~, u’. E., in “The Proteins” (H. Neurath and K. Bailey, eds.), Vol. II, Part A, p. 355. Bcademic Press, New York, 1954. SUJINER, J. B., AXD SOMERS, G. F., “Chemistry and Methods of Enzymes,” p. 24. Academic Press, ?;em Tork, 1947. BJERHUX, J., SCHWARZENBACH, G., ASD SILLIN, L. G., Compilers, “Stability Constants of Metal-Ion Complexes,” Part I : “Organic Ligands,” p. 76. Special Publ. No. 6. The Chemical Society, London, 1959. HAWKE, J. C., Biochem. J. 64, 311 (1956). HUGHER, A., Biochem. J. 29, 437 (1935). DELEZENNE. C., AND FOURNEAU, E., BUM/. sot. c-him. 15, 421 (1914).
14. S~USDERS,
15. 16.
17.
18.
19. 20. 21.
OF PHOSPHOLIPASE
ON LECITHINS
379
22. MCBAIN, J. W., “Colloid Science,” pp. 244-251. D. C. Heath and Co., Boston, Mass., 1950. E., “Solu23. MCBAIN, M. E. L., .~ND HUTCHINSON, bilization,” pp. 28-43. Academic Press, New York, 1955. 24. JIRGENSOXS B., “Organic Colloids,” p. 60. Elsevier P;bl. Co., Amsterdam, 1958. 25. KLEVEIYS, H. B., J. Phys. & Colloid Chem. 52, 130 (1948). 26. EHRESBERG, A., Acta Chem. Stand. 11, 1257 (1957). 27. GOSTIX~. L. J.. .~XD MORRIS, M. S., J. Am. Chwn. Sot. 71, 1998 (1949). 28. GOSTINC, L. J., Advances in Protein Chem. 11, 429 (1956). 29. SAUNDERS. L., AXD THOMAS, I. L., J. Chem. sot. 1958, 483. in Bio30. SCHACHX~Y, H. K., “Ultracentrifugation chemistry.” Academic Press, New York, 1959.