- Email: [email protected]
Effects of crotoxin (lecithinase A) on egg yolk and yolk constituents
Effects of Crotoxin (Lecithinase A) on Egg Yolk and Yolk Constituents Robert E. Feeney, L. R. MacDonnell and Heinz Fraenkel-Conrat’ Laboratory,=Albany, California ReceivedJuly 7, 1953
From the Western Regional Research
INTRODUCTION
Egg yolk is a stable emulsion containing approximately 50 % solids. It is an oil-in-water type of dispersion, which is stabilized by high contents of cholesterol, phospholipides, and lipoproteins. The phospholipide constituents are principally lecithin and cephalin and are present in the approximate amounts of 14 and 5 Y. respectively, on a dry-weight basis. In connection with investigations of the role of the yolk constituents in the emulsification properties of egg yolk and changes therein caused by heating or freezing (l), we studied the effects of various physical and chemical treatments on the properties of egg yolk. One approach to the problem was the use of a specific enzyme, a lecithinase, which would modify the important phospholipide constituents. Crystalline crotoxin (2), the neurotoxin from the venom of the rattlesnake, Crotalus t. terrij&s, has been employed for the following reasons: (a) It was available in a purified crystalline form, and (b) it is a lecithinase A, an esterase that splits off an unsaturated fatty acid from lecithin (and sometimes from cephalin), producing a desoleophospholipide (3-5). Such a reaction should change the emulsification or solubility characteristics of yolk without splitting the lipoprotein complexes as might occur by hydrolysis between the phosphorus and the glycerol or amine. In the present study, egg yolk, isolated phospholipides, and lipoproteins were treated with crystalline crotoxin, and certain of their properties have been studied. * Present address:The Virus Laboratory, University of California, Berkeley, Calif. 2 Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 130
mTm(:TS OF’ CRoTr)Srs
I3 1
The yolk phospholipides were partially purified preparations obtained by the conventional methods of diethyl ether extra&ion of lyophilized yolk in a Soxhlet apparatus followed by precipitation of the crude mixed phospholipides with acetone. Lecithin and cephalin were separated by means of their differential soluhilities in ethanol. Analyses on Prepn. 4-B (crude phospholipides) were: P, 3.970; N, 1.9%; and amino N, 0.64% (34Tc of iota1 N). Analyses on Prepn. 16 (lecit,hin) mere: l’, 3.9%; X, 1.870. The lipoproteins were prepared from the yolk of fresh eggs by the procedures of Alderton and Fevold (6) and Fevold and Lausten (7) for the preparations of lipovitellin and lipovitellenin, respectively. Preparation 11 was a crude preparntion of lipovitellin obtained by a single dialysis and diethyl ether extraction of :t Sharplcs centrifugate. Preparation 8-l was an aliquot of 21 which had been sub jetted to two further successive dialyses, lyophilizations, and ether extractions. It contained 14% lipide. Preparation B was extracted several times with ether, but was not dialyzed between extractions. Preparations C-l and C-2, lipovitellin and lipovitellenin, respectively, were subjected to repeated dialyses and ether extractions. They contain 13.4 and 10.87n N and 10 and 29y0 lipide, respectively. The lipide-free residues, vitellin and vitellenin, contained 15.3 and lS.570 N ant1 1.54 and 0.59% P, respective1y.3 The crystalline crotoxin was prepared according to Slotta and Fraenkel-Conrat (2). Toxic activities were confirmed by mouse-inoculation tests. Crotoxin treatments were done at 37°C. unless otherwise indicated. Phospholipide preparations were in the form of aqueous suspensions, and lipoproteins in 512% sodium chloride solutions. For the routine hemolytic tests, oxalated rat blood was obtained by decapitation. The cells were centrifuged, washed twice with 2-10 vol. of 0.9% sodium chloride, and stored in 10-20 vol. of the saline at 4°C. The cell suspension was diluted with saline approximately 209fold on a cell-volume basis for the hemolytic t,ests (so as to give a calorimeter reading of i5-85 in a Klett-Summerson photoelectric calorimeter equipped with a 660 rnp filter). In the performance of a hemolytic test, 0.02-0.1 ml. of a solution or dispersion of the test material was added to the calorimeter tube. Five milliliters of the blood-cell suspension was pipetted rapidly into the tube, and calorimeter readings were usually recorded after 2, 4, 7: 10, and 20 min. at room temperature. Hemolytic activities were expressed in terms of a preparation of lysophospholipide from yolk, designated 21-C-12. The properties of this preparation and accuracy of the assays are described below. The rabbit blood for comparative studies was obtained by auricular venipuncture. 3 At least part of the phosphorus of the vitellin and vitellenin preparations may be due to contamination with phosvitin, the phosphorus-rich protein of egg yolk (8).
132
FEENEY, Ma4CDONNELL A4ND FR4ENKEL-CONRAT RESULTS
Properties of Crotoxin-Treated
Yolk
Yolk incubated with 1 or 10 kg. crotoxin/ml. evidenced no important changes in appearance, but extensive changes in physical and biochemical properties were found. The lipoprotein, lipovitellin, was not precipitated upon dilution of treated yolk with several volumes of water, which is in contrast to its ready precipitation from normal yolk. A very marked
Properties
TABLE I of Fractions from Crotoxin-Treated
Fraction
Description’
NO. __--.
21-o 21-A 21-B 21-c 21-C-12
Unfratitionated yolk Acetone filtrate (aqueous) Ether extract of residue Alcohol extract of residue Ether ppt. of 21-C
Yolk Properties
ROXAmount for very of hem. testb original activity’ ~--~--~ rg.lml. %
Molar ratio P/N
-I 10 3
35 32
--
:0.01 0.008 3.80 1.89 5.38 2.800
0.91 0.87
a Details are given in text. b As determined by method described in text. Blood was washed rat blood cells diluted approximately 350-fold on whole blood basis in 0.9% saline. c Total activity of fractions was as high as 125% of original activity (21-A plus 21-C). d Activity determined on a liquid volume basis. 8 Amino N content was 0.82%.
reduction occurred in the amount of gelation caused by freezing, and a strong hemolytic activity was produced (Table I). The details of one experiment, in which crotoxin reduced the gelation when the enzymatic action was allowed to occur either before or after freezing, follow: 750 ml. of blended yolk containing 0.1% Merthiolate was divided into five 150-ml. aliquots and either 1 ml. of a crotoxin solution or 1 ml. of water was blended with each aliquot to give the following samples: a, 10 pg./ml. crotoxin; b, 10 pg./ml. crotoxin; c, 1.0 pg./ml. crotoxin; and cl and e, no added enzyme. Samples a and d were immediately frozen at -7O”C., then after 30 min. transferred to -17°C. for 48 hr., thawed, and finally incubated for 48 hr. at 37°C. Samples b, c, and
ISFFECTS
OF
C!lZOTOXIN
1x3
c were treat’ed similarly with the exception that they were incubated prior to freezing instead of afterwards. Samples a, d, and e all exhibited the typical stiff gel immediat,ely on thawing, whereas samples b and G were sufficiently fluid to flow from their containers. When a and d were incubated, the viscosity of a reduced until it was grossly similar to 0 and c. Thus, orotoxin not only markedly prevented the gelation caused by freeziiig when it] was allowed to act before freezing, but it also reduced the gelatiotl after it was already formed. Comparative viscosities, made with a torsion viscometer employing a piano wire--knife blade arrangemelit (9), showed the crotoxin-treated yolks to have 10-20~~ of the amount of gelation as the untreated. Preparation
and General Properties of Lysophospholipides
from
Yolk
Lysophosphat.ides were prepared by crot,oxin treatment of yolk, lipoprotein preparations, and purified phospholipides. In one large preparation from yolk, 5 1. was blended with 2.5 g. Merthiolate dispersed in 25 ml. water and 5.0 mg. erotoxin dissolved in 5.0 ml. dilute phosphate buffer. The mixture was incubated at 37°C. Although hemolytic assays (see below) showed approximately twice the concentration of lysophospholipides at 48 hr. as compared to 24 hr., incubation was discontinued at 48 hr., because the initiation of microbial spoilage was evident. One liter of the incubated mixture was fractionated as follows (see Table I) : The yolk was diluted with 500 ml. water and mixed with 2 1. acetone, which precipitated considerable insoluble material. After the mixture had stood overnight at room temperature, 11. more of acetone was added. The precipitat,e was removed by filtration and washed with 500 ml. acetone followed by 500 ml. diethyl ether. The filtrate (21-A), containing considerable organic matter, separated into two phases after 6 days in the refrigerator. Although it contained most of the hemolyt,ic activity, it was not fractionated because of the greater facility with which the precipitate could be handled. The precipiate was extracted by stirring for 1 hr. with 11. ether (Prepn. 21-B) and the residue further extracted with 1 1. of 95 % ethyl alcohol. The extract (Prepn. 21-C) contained approximately 30 y0 of t,he original hemolytic activity, and the activity could be precipitated essentially quantitatively by dilution with 10-20 vol. ether and storage at - 17°C. for 12-16 hr. Preparation 21-C-12 was obtained by precipitation with 10 vol. ether, recrystallization two times from absolut]e alcohol, and filtration at - 17°C. (Table I). It was obtained in crystalline or “semicrystalline” form when recrystallized from alcohol at
134
FEENEY,
MACDONNELL
AND
FRAENKEL-CONRAT
- 17’C.’ As evidenced by the nitrogen and phosphorus analyses and the aolubilitiea in diethyl ether and ethanol, it was apparently a mixture of lyaocephalin, lyaolecithin, and a small amount of material low in phoaphorua. These analyses agreed closely with those of Chargaff and Cohen (10). Several attempts at further purification were unsuccessful, and preparations with the purities reported by Levene, Rolf, and Simma (11) were not obtained. Action of Crotoxin on Yolk Phospholipides
Strong hemolytic activity was obtained by crotbxin treatment of buffered emulsions of crude mixed yolk phoapholipidea or partially purified lecithin preparations. Concomitant with the development of hemolytic activity, partial clearing or solubilization of the emulsions occurred. This aolubilization was greatly increased when the emulsions were made alkaline with dilute sodium hydroxide. It was nearly complete with the lecithin preparations but only partially so with the crude mixed phoapholipidea. Although emulsions of lecithin were usually rapidly attacked by crotoxin, large variations in the rates and extents of action were encountered. For example, in one series of experiments with 1.0 mg./ml, lecithin and a relatively high amount of crotoxin, 25 pg./ml., conversions ranging from less than 5 % to greater than 50 % were obtained in 5 hr. at 37°C. In contrast to the results with lecithin and the crude mixed phoapholipides, a cephalin preparation was not attacked by the crotoxin. Treatment with crotoxin did not produce hemolytic activity, clearing of the emulsion with alkali, or insolubility in diethyl ether. Action of Crotoxin on Yolk Li-poproteins
Incubation of crotoxin with dispersions of lipovitellin or lipovitallenin preparations in 5-10 $!&sodium chloride gave products that were hemolytic, yielded hemolytically active ethanol extracts, and were highly aoluble in distilled water. The hemolytic activities obtained in five different experiments with three preparations of lipovitellin and a preparation of lipovitellenin are listed in Table II. The highest yields of hemolytic activity were obtained with the preparations of lipovitellenin. In Expts. 4 The material microscopically resembled that described by Delezenne and Fourneau (3) and separated as small rounded hemispheres with nearly perfect edges and a bright brilliance. Upon removal of the material from the cold room, however, it became amorphous.
EFFECTS
OF (‘ROTOXIU
13.5
\:l and L’LI the yields of lysolecithin with lipovitc4lenin were 41 and 55 “/b, respectively. Tile efl’ects of varying the condit,ions of digest,ion were studied in t,he experiments of Table II. In an extension of Expt. II, the following acTABLE Crotoxin
II
Treatment
of Lipoproteins hhterials”
Protein
I
Vitellin
A ~~ --~~
II-a II-6 II-C 11-d
Vitellin” (II-a)< (II-b)c (II-c)c
B 1 B B
III IV
Vitellin Vitellin ~_._~_~~~
A ~ A j ~- ~-
V-a
V-b
Vitellin (V-a)*
VI-a VI-b
Vitellenin (VI-u)*
VII
Vitellenin
~--”
Apgarent Time incubated lysolecithin” CR- Sodium Diethyltoxin chloride ether 1
4.0 ~ 5
0
48
07 ,o 5.8
.-
loo loo 100 100
0.8
0
s
1.2 1.6 2.0
0 0 0
x 8 8
24 48 -9 i” 96
01 0.9 2.2 2 2
33 100
0.7 0.7
11 11
5 5
72 72
3.1 2.0
-
;--.
.-.
18 18
18. 21.
5: 5
0 0
24 48
1.9 C1.9’
c-2
18 18
18. 21.
5 5
0 0
24 48
4.3 8.1
~ C-2
43
14.
10
5
48
j ::: ~~ j
c-2
11.
a All experiments were performed with pH 6X-7.0 phosphate buffer (0.02-0.05 The temperature of incubation was 37°C. b Apparent lysolecithin determined as described in text by hemolytic assay and calculated on basis of activity of Prepn. 21-C-12. c This was a comparatively large-scale preparation, 1200 ml. containing 120 g. lipovitellin. After the initial 24 hr. incubation, the dispersion was held at room temperature under water-pump vacuum for 30 min. to remove part of ether. 21 further additon of enzyme (0.4 lg./ml.) was then made and incubations continued for 24 hr. more (II-b). 11-c and 11-d IT-ere obtained by adding more enzyme and incubating for 24 hr. more in each case. d V-b and VI-b m-ere obtained by adding more enzyme and incubating for a further 24 hr. V-a and VI-e, respectively. a Assay showed a definite decrease in activity as compared to IV-n. Preparation, however, precipitated under assay conditions. M).
I36
FEENEY,
MACDONNELL
AND
FRAENKEL-CONRAT
tivities (mg./ml.) were obtained when aliquots of 11-a (after ether removal) were incubated for 24 hr. under the conditions indicated: No additions or changes,0.8; plus 2.0 pg./ml. crotoxin, 0.8; plus 20.0 pg./ml. crotoxin, 1.5; plus 2.0 pg./ml. crotoxin and an ash of yeast extract equivalent to 20 mg./ml. of t,he yeast extract, 1.0; plus 2.0 pg./ml. crotoxin and a salt mixture,6 1.1; plus 2.0 pg./ml. crotoxin and dilution five times with 5 y0 sodium chloride, 1.8; and plus 20.0 pg./ml. crotoxin and dilution five times with 5 Y0sodium chloride, 2.2. In other experiments no effect was given by the addition of 0.002 M tricalcium citrate or 0.002 M calcium chloride. In contrast to the very low water solubility of lipovitellin and lipovitellenin, the crotoxin-treated lipoproteins readily dissolved in distilled water to give a 5 % solution. In the presenceof low and high concentrations of salt, however, their solubilities were more similar to those of the original proteins, i.e., insoluble in low (0.1-0.2 %) concentrations of salt but soluble in high concentrations. Although water solutions of crotoxintreated lipovitellin (1 X) were rapidly precipitated at 95-lOO”C., solutions in 5 $&salt did not precipitate after 10 min. Controls containing untreated protein in 5 Y0salt precipitated in less than 1 min. at 95-100°C. The phospholipides did not appear to be split from the lipoprotein complexes by crotoxin treatment. A serious problem in this interpretation was the very low solubility of the lysophospholipides in diethyl ether, which is empirically used as a solvent to remove “unbound” lipides from lipoproteins. However, partial precipitation from solution by salt or pH adjustment did not reduce the lysophospholipide content, and two attempts to differentially extract the lyso compounds with ethanol at low temperatures were unsuccessful. Ethanol extraction at room temperature removed the phospholipide, and highly active lysophospholipide preparations were obtained by ether precipitation of the ethanol extract. Hewdytic
Activities and Assays
Hemolysis was characterized by the low concentrations of lysophospholipides necessary and by the partial “all or none” or threshold requirements. The turbidity readings obtained in one experiment are plotted against the lysolecithin concentrations in Fig. 1. The typical “all 6 The salt mixture gave the following cations as the hydrochlorides in the concentrations (p.p.m.) indicated: K+, 10; Mg++, 2.5; Zn”, 0.25; Fe+++, 0.25; Mn++. 0.25; CO+++, 0.10; and Cu++, 0.10.
13;
-iCFFEC'TS OF CIEO'I'OXTN
or noLlc’~ or threshold characteristic is evident ill this ligure ill ,\vhich 1.-k ps./rnl. ga\-e only slight hemolysis while approximateI>, t\vic’r this cnolic*rntration (3.0 pg./ml.) gave estelisi\.e hcmolysis. The reproducibility of the qnantitati\e relationships was found to t)c depe&ent upon the blood sample employed, the method of dispersion of the lysophospholipide in preparation for assay, possibly the age of this dispersion, and unidentified factors. Nevertheless, a rather high 1
I
I
I
I
I
I 2
I 4
I 6
I 8
I IO
TIME FIG.
figures
IN MINUTES
AFTER
‘W
ADDITIONS
1. Hemolyses with graduated levels of lysophospholipide. Kumeric:J on curves refer to pg./ml. lysophospholipide (Prepn. 21-(1-12) added.
reproducibility was found in a series of 27 experiments with a total of 97 tests of the standard preparation 21-C-12. As compared on the basis of the percentages of tests at different concentrations requiring less than 20 min. for 70 % hemolysis, the following were the resulk obtained: 1.0-1.4 pg./ml., 0% (0 of 4); l-7-2.0 pg./ml., 25% (6 of 24); 2.3%2.i pg./ml., 76% (32 of 42); and 3.0-3.3 pg./ml., 89% (24 of 27). The preparation of samples was found partJic*ularly important. Apparently, the critical factor was the formation of a finely dispersed suspension or solution which was routinely accomplished by diluting ti ii-10 mg./ml. et,hanol solution lo- to 20-fold with water. This stock
138
FEENEY,
MACDONNELL
bND
FRAENKEL-CONRBT
suspension was usually diluted 3-6 times prior to assay, and 0.02-0.1 ml. was added per tube of 5 ml. of blood cell suspension. Although small amounts of ethanol were demonstrated to influence the hemolysis, the effects of the employed concentrations were small, and both standards and samples under assay were prepared similarly. A second way of preparation was solution by the addition of 0.1 N sodium hydroxide, with or without the addition of ethanol. The use of alkali, however, caused inactivation of some samples and was therefore not routinely employed. No direct hemolytic effect was exerted by the crotoxin in concentrations equivalent to those used in the hydrolyses, and it was therefore unnecessary to inactivate the enzyme prior to assay. The least accurate assays were obtained with whole yolk, crude yolk fractions, and the lipoproteins. Part of the difficulty with the lipoproteins was precipitation during the assay. This was a result of their unique solubilities; i.e., they were soluble in distilled water and strong salt but relatively insoluble in the dilute salt solution necessary for suspension of the blood cells. Although the possibility of precipitation made it necessary to assay the lipoproteins at mult,iple levels, it was usually possible to find a series of concentrations that gave slopes similar to the standard.
DISCUSSION Although phospholipides and their complexes are of major importance in the biophysical and biochemical systems of living materials, their actual roles are only very poorly understood. The present studies with crotoxin-treated lipoproteins and frozen and thawed egg yolk support the long-advanced hypothesis that the gelation of yolk on freezing and thawing is related to changes involving the lipoproteins (1, 7, 12). These results agree with the older studies of Tressler (13) in which gelation was reduced by treatment with the proteolytic enzyme pepsin or with crude enzymes. Other studies at this laboratory which will be reported elsewhere give still further data indicating the importance of the lipoproteins in this freezing-induced deterioration, as well as their roles in those properties of yolk which are responsible for heat coagulation. The importance of the phospholipides in the gross properties of yolk has also been indicated by studies with microorganisms. In such studies, precipitates were observed in media containing egg yolk and inoculated with members of the genus Clostridium (14) and certain Bacilli (15).
EFFECTS
OF
CROTOXIN
13!1
The Bacilli mere also shown to attack phospholipide preparations (15). However, only the lecithinase of t!he clostridia has been characterized, and this as lecithinase C (4, 14). Lecithinase C is a phosphatase and cleaves the phospholipide molecule bet’ween the phosphate and the glycerol components. The data suggested that crotoxin attacked the lipoprotein complexes directly without splitting the lipide from the protein, but conclusive proof of such action was not obtained. This was a result of the empirical criteria for defining a lipoprotein. The definition as usually employed defines that lipide, which is bound to protein to form a lipoprotein, is not extra,cted by the more nonpolar solvent,s such as petroleum ether or diethyl ether. The insolubility of the lysophospholipides in solvents of this nature makes it difficult to apply such an empirical formula. ,1 number of important problems still present themselves with regard to enzymatic action of crotoxin on the yolk constituents. Foremost among t,hese are the variable rates of reaction obt’ained and apparent lack of activity on partially purified cephalin. This activity of the enzyme on cephalin in unfract’ionated yolk and the absence of activit,y on the purified material confirm the observations of Chargaff and Cohen ilO), who post,ulated the possible presence of susceptible complexes of cephalin ill the intact yolk. Of interest in this regard are the many studies on t,he action of the lecit’hinase containing venoms of different snakes on erythrocytes (16). It is possible that the reason why certain venoms do not act upon erythrocytes concerns their capability of hydrolyzing cephalin. With regard to egg yolk it is important t.o determine whet,her or not t,here is an enzymatically susceptible complex of cephalin in egg yolk and consequently whether or not the isolated cephalin is an artifact. So at,tempt was made in these studies to refine the hemolytic assay developed. Of the various factors discussed in the text, the most important appeared to be the state of lysophospholipide in the dispersions employed for assay. It should be possible, by better standardization of sample preparations, to employ the hemolytic assay for preparations t,hat do not precipitate on dilution in water or salt. The fact that au “all or none” characteristic or threshold amount is necessary for hemolysis might indicate a reaction with, or absorption by, the erythrocyte or ils constituents prior to the occurrence of hnmolysis. Surface-tcnsioll rcdilctiorl ( 13 is also probably a factor.
140
FEENEY, MACDONNELL
AND
FRAENKEL-CONRAT
SUMMARY
The gelation of egg yolk caused by freezing was reduced by incubation with crotoxin (lecithinase A) before or after freezing. The lipoprotein fractions of yolk, lipovitellin and lipovitellenin, were attacked by crotoxin. The lysophospholipoproteins formed were hemolytic, had changed solubilities in water, and could be split by ethanol to yield impure lysophospholipide (desoleophospholipide) and the protein moiety. The lipoprotein complex did not appear split by action of the enzyme. The possible relationships between the changed properties of the lipoproteins and the modified gelation characteristics of frozen yolk were discussed. The hemolytic activities of yolk fractions treated with crotoxin were studied. With purified lysophospholipide preparations hemolysis of rat and rabbit erythrocytes exhibited a partial “all or none” characteristic. REFERENCES 1. URBAIS, O., AND MILLER, J., Znd. Eng. Chem. 22, 355 (1930). 2. SLOTTA, K. H., AND FRAENKEL-CONRAT, H. L., Ber. 71B21076(1938). 3. DELEZENNE,~., ANL)FOURNEAU,E., BulZ.soc.chim.[4] 16,421 (1914). 4. CELMER, W. D., AND CARTER, H. E., Phgsiol. Revs. 32, 167(1952). 5. PORGES, N., Science 117, 47 (1953). 6. ALDERTON, G., AND FEVOLD, H. L., Arch. Biochem. 8, 415(1945). 7. FEVOLD, H. L., AND LAUSTEN, A., Arch. Biochem. 11,1 (1946). 8. MECHAM, D. K., AND OLCOTT,H. S.,J. Am. Chem. Sot. 71,367O (1949). 9. OWENS, H., Food Znds. 19, 606 (1947). 10. CHARGAFF, E., AND COHEN, S. S., J. Biol. Chem. 129, 619 (1939). 11. LEVENE, I?. A., ROLF, I. P., AND SIMMS, H. S., J. Biol. Chem. 68,859 (1924). 12. LEA, C. H., AND HAWHE, J. C., Biochem. J. (London) 62,105 (1952). 13. TRESSLER, S. K., U. S. Patent 1,870,269 (1932). 14. MCCLUNG, L. S.,HEIDENREICH, P., AND TOABI, R., J. Bacterial. 61, 751 (1946). 15. COLMER, A. R., J. Bacterial. 66, 777 (1948). 16. ROY, A. C., Nature 166,696(1945). 17. PETHICA, B. A., AND SCHULMAN,J. H., Biochem. J. (London) 63,177 (1953).
From the Western Regional Research
INTRODUCTION
Egg yolk is a stable emulsion containing approximately 50 % solids. It is an oil-in-water type of dispersion, which is stabilized by high contents of cholesterol, phospholipides, and lipoproteins. The phospholipide constituents are principally lecithin and cephalin and are present in the approximate amounts of 14 and 5 Y. respectively, on a dry-weight basis. In connection with investigations of the role of the yolk constituents in the emulsification properties of egg yolk and changes therein caused by heating or freezing (l), we studied the effects of various physical and chemical treatments on the properties of egg yolk. One approach to the problem was the use of a specific enzyme, a lecithinase, which would modify the important phospholipide constituents. Crystalline crotoxin (2), the neurotoxin from the venom of the rattlesnake, Crotalus t. terrij&s, has been employed for the following reasons: (a) It was available in a purified crystalline form, and (b) it is a lecithinase A, an esterase that splits off an unsaturated fatty acid from lecithin (and sometimes from cephalin), producing a desoleophospholipide (3-5). Such a reaction should change the emulsification or solubility characteristics of yolk without splitting the lipoprotein complexes as might occur by hydrolysis between the phosphorus and the glycerol or amine. In the present study, egg yolk, isolated phospholipides, and lipoproteins were treated with crystalline crotoxin, and certain of their properties have been studied. * Present address:The Virus Laboratory, University of California, Berkeley, Calif. 2 Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture. 130
mTm(:TS OF’ CRoTr)Srs
I3 1
The yolk phospholipides were partially purified preparations obtained by the conventional methods of diethyl ether extra&ion of lyophilized yolk in a Soxhlet apparatus followed by precipitation of the crude mixed phospholipides with acetone. Lecithin and cephalin were separated by means of their differential soluhilities in ethanol. Analyses on Prepn. 4-B (crude phospholipides) were: P, 3.970; N, 1.9%; and amino N, 0.64% (34Tc of iota1 N). Analyses on Prepn. 16 (lecit,hin) mere: l’, 3.9%; X, 1.870. The lipoproteins were prepared from the yolk of fresh eggs by the procedures of Alderton and Fevold (6) and Fevold and Lausten (7) for the preparations of lipovitellin and lipovitellenin, respectively. Preparation 11 was a crude preparntion of lipovitellin obtained by a single dialysis and diethyl ether extraction of :t Sharplcs centrifugate. Preparation 8-l was an aliquot of 21 which had been sub jetted to two further successive dialyses, lyophilizations, and ether extractions. It contained 14% lipide. Preparation B was extracted several times with ether, but was not dialyzed between extractions. Preparations C-l and C-2, lipovitellin and lipovitellenin, respectively, were subjected to repeated dialyses and ether extractions. They contain 13.4 and 10.87n N and 10 and 29y0 lipide, respectively. The lipide-free residues, vitellin and vitellenin, contained 15.3 and lS.570 N ant1 1.54 and 0.59% P, respective1y.3 The crystalline crotoxin was prepared according to Slotta and Fraenkel-Conrat (2). Toxic activities were confirmed by mouse-inoculation tests. Crotoxin treatments were done at 37°C. unless otherwise indicated. Phospholipide preparations were in the form of aqueous suspensions, and lipoproteins in 512% sodium chloride solutions. For the routine hemolytic tests, oxalated rat blood was obtained by decapitation. The cells were centrifuged, washed twice with 2-10 vol. of 0.9% sodium chloride, and stored in 10-20 vol. of the saline at 4°C. The cell suspension was diluted with saline approximately 209fold on a cell-volume basis for the hemolytic t,ests (so as to give a calorimeter reading of i5-85 in a Klett-Summerson photoelectric calorimeter equipped with a 660 rnp filter). In the performance of a hemolytic test, 0.02-0.1 ml. of a solution or dispersion of the test material was added to the calorimeter tube. Five milliliters of the blood-cell suspension was pipetted rapidly into the tube, and calorimeter readings were usually recorded after 2, 4, 7: 10, and 20 min. at room temperature. Hemolytic activities were expressed in terms of a preparation of lysophospholipide from yolk, designated 21-C-12. The properties of this preparation and accuracy of the assays are described below. The rabbit blood for comparative studies was obtained by auricular venipuncture. 3 At least part of the phosphorus of the vitellin and vitellenin preparations may be due to contamination with phosvitin, the phosphorus-rich protein of egg yolk (8).
132
FEENEY, Ma4CDONNELL A4ND FR4ENKEL-CONRAT RESULTS
Properties of Crotoxin-Treated
Yolk
Yolk incubated with 1 or 10 kg. crotoxin/ml. evidenced no important changes in appearance, but extensive changes in physical and biochemical properties were found. The lipoprotein, lipovitellin, was not precipitated upon dilution of treated yolk with several volumes of water, which is in contrast to its ready precipitation from normal yolk. A very marked
Properties
TABLE I of Fractions from Crotoxin-Treated
Fraction
Description’
NO. __--.
21-o 21-A 21-B 21-c 21-C-12
Unfratitionated yolk Acetone filtrate (aqueous) Ether extract of residue Alcohol extract of residue Ether ppt. of 21-C
Yolk Properties
ROXAmount for very of hem. testb original activity’ ~--~--~ rg.lml. %
Molar ratio P/N
-I 10 3
35 32
--
:0.01 0.008 3.80 1.89 5.38 2.800
0.91 0.87
a Details are given in text. b As determined by method described in text. Blood was washed rat blood cells diluted approximately 350-fold on whole blood basis in 0.9% saline. c Total activity of fractions was as high as 125% of original activity (21-A plus 21-C). d Activity determined on a liquid volume basis. 8 Amino N content was 0.82%.
reduction occurred in the amount of gelation caused by freezing, and a strong hemolytic activity was produced (Table I). The details of one experiment, in which crotoxin reduced the gelation when the enzymatic action was allowed to occur either before or after freezing, follow: 750 ml. of blended yolk containing 0.1% Merthiolate was divided into five 150-ml. aliquots and either 1 ml. of a crotoxin solution or 1 ml. of water was blended with each aliquot to give the following samples: a, 10 pg./ml. crotoxin; b, 10 pg./ml. crotoxin; c, 1.0 pg./ml. crotoxin; and cl and e, no added enzyme. Samples a and d were immediately frozen at -7O”C., then after 30 min. transferred to -17°C. for 48 hr., thawed, and finally incubated for 48 hr. at 37°C. Samples b, c, and
ISFFECTS
OF
C!lZOTOXIN
1x3
c were treat’ed similarly with the exception that they were incubated prior to freezing instead of afterwards. Samples a, d, and e all exhibited the typical stiff gel immediat,ely on thawing, whereas samples b and G were sufficiently fluid to flow from their containers. When a and d were incubated, the viscosity of a reduced until it was grossly similar to 0 and c. Thus, orotoxin not only markedly prevented the gelation caused by freeziiig when it] was allowed to act before freezing, but it also reduced the gelatiotl after it was already formed. Comparative viscosities, made with a torsion viscometer employing a piano wire--knife blade arrangemelit (9), showed the crotoxin-treated yolks to have 10-20~~ of the amount of gelation as the untreated. Preparation
and General Properties of Lysophospholipides
from
Yolk
Lysophosphat.ides were prepared by crot,oxin treatment of yolk, lipoprotein preparations, and purified phospholipides. In one large preparation from yolk, 5 1. was blended with 2.5 g. Merthiolate dispersed in 25 ml. water and 5.0 mg. erotoxin dissolved in 5.0 ml. dilute phosphate buffer. The mixture was incubated at 37°C. Although hemolytic assays (see below) showed approximately twice the concentration of lysophospholipides at 48 hr. as compared to 24 hr., incubation was discontinued at 48 hr., because the initiation of microbial spoilage was evident. One liter of the incubated mixture was fractionated as follows (see Table I) : The yolk was diluted with 500 ml. water and mixed with 2 1. acetone, which precipitated considerable insoluble material. After the mixture had stood overnight at room temperature, 11. more of acetone was added. The precipitat,e was removed by filtration and washed with 500 ml. acetone followed by 500 ml. diethyl ether. The filtrate (21-A), containing considerable organic matter, separated into two phases after 6 days in the refrigerator. Although it contained most of the hemolyt,ic activity, it was not fractionated because of the greater facility with which the precipitate could be handled. The precipiate was extracted by stirring for 1 hr. with 11. ether (Prepn. 21-B) and the residue further extracted with 1 1. of 95 % ethyl alcohol. The extract (Prepn. 21-C) contained approximately 30 y0 of t,he original hemolytic activity, and the activity could be precipitated essentially quantitatively by dilution with 10-20 vol. ether and storage at - 17°C. for 12-16 hr. Preparation 21-C-12 was obtained by precipitation with 10 vol. ether, recrystallization two times from absolut]e alcohol, and filtration at - 17°C. (Table I). It was obtained in crystalline or “semicrystalline” form when recrystallized from alcohol at
134
FEENEY,
MACDONNELL
AND
FRAENKEL-CONRAT
- 17’C.’ As evidenced by the nitrogen and phosphorus analyses and the aolubilitiea in diethyl ether and ethanol, it was apparently a mixture of lyaocephalin, lyaolecithin, and a small amount of material low in phoaphorua. These analyses agreed closely with those of Chargaff and Cohen (10). Several attempts at further purification were unsuccessful, and preparations with the purities reported by Levene, Rolf, and Simma (11) were not obtained. Action of Crotoxin on Yolk Phospholipides
Strong hemolytic activity was obtained by crotbxin treatment of buffered emulsions of crude mixed yolk phoapholipidea or partially purified lecithin preparations. Concomitant with the development of hemolytic activity, partial clearing or solubilization of the emulsions occurred. This aolubilization was greatly increased when the emulsions were made alkaline with dilute sodium hydroxide. It was nearly complete with the lecithin preparations but only partially so with the crude mixed phoapholipidea. Although emulsions of lecithin were usually rapidly attacked by crotoxin, large variations in the rates and extents of action were encountered. For example, in one series of experiments with 1.0 mg./ml, lecithin and a relatively high amount of crotoxin, 25 pg./ml., conversions ranging from less than 5 % to greater than 50 % were obtained in 5 hr. at 37°C. In contrast to the results with lecithin and the crude mixed phoapholipides, a cephalin preparation was not attacked by the crotoxin. Treatment with crotoxin did not produce hemolytic activity, clearing of the emulsion with alkali, or insolubility in diethyl ether. Action of Crotoxin on Yolk Li-poproteins
Incubation of crotoxin with dispersions of lipovitellin or lipovitallenin preparations in 5-10 $!&sodium chloride gave products that were hemolytic, yielded hemolytically active ethanol extracts, and were highly aoluble in distilled water. The hemolytic activities obtained in five different experiments with three preparations of lipovitellin and a preparation of lipovitellenin are listed in Table II. The highest yields of hemolytic activity were obtained with the preparations of lipovitellenin. In Expts. 4 The material microscopically resembled that described by Delezenne and Fourneau (3) and separated as small rounded hemispheres with nearly perfect edges and a bright brilliance. Upon removal of the material from the cold room, however, it became amorphous.
EFFECTS
OF (‘ROTOXIU
13.5
\:l and L’LI the yields of lysolecithin with lipovitc4lenin were 41 and 55 “/b, respectively. Tile efl’ects of varying the condit,ions of digest,ion were studied in t,he experiments of Table II. In an extension of Expt. II, the following acTABLE Crotoxin
II
Treatment
of Lipoproteins hhterials”
Protein
I
Vitellin
A ~~ --~~
II-a II-6 II-C 11-d
Vitellin” (II-a)< (II-b)c (II-c)c
B 1 B B
III IV
Vitellin Vitellin ~_._~_~~~
A ~ A j ~- ~-
V-a
V-b
Vitellin (V-a)*
VI-a VI-b
Vitellenin (VI-u)*
VII
Vitellenin
~--”
Apgarent Time incubated lysolecithin” CR- Sodium Diethyltoxin chloride ether 1
4.0 ~ 5
0
48
07 ,o 5.8
.-
loo loo 100 100
0.8
0
s
1.2 1.6 2.0
0 0 0
x 8 8
24 48 -9 i” 96
01 0.9 2.2 2 2
33 100
0.7 0.7
11 11
5 5
72 72
3.1 2.0
-
;--.
.-.
18 18
18. 21.
5: 5
0 0
24 48
1.9 C1.9’
c-2
18 18
18. 21.
5 5
0 0
24 48
4.3 8.1
~ C-2
43
14.
10
5
48
j ::: ~~ j
c-2
11.
a All experiments were performed with pH 6X-7.0 phosphate buffer (0.02-0.05 The temperature of incubation was 37°C. b Apparent lysolecithin determined as described in text by hemolytic assay and calculated on basis of activity of Prepn. 21-C-12. c This was a comparatively large-scale preparation, 1200 ml. containing 120 g. lipovitellin. After the initial 24 hr. incubation, the dispersion was held at room temperature under water-pump vacuum for 30 min. to remove part of ether. 21 further additon of enzyme (0.4 lg./ml.) was then made and incubations continued for 24 hr. more (II-b). 11-c and 11-d IT-ere obtained by adding more enzyme and incubating for 24 hr. more in each case. d V-b and VI-b m-ere obtained by adding more enzyme and incubating for a further 24 hr. V-a and VI-e, respectively. a Assay showed a definite decrease in activity as compared to IV-n. Preparation, however, precipitated under assay conditions. M).
I36
FEENEY,
MACDONNELL
AND
FRAENKEL-CONRAT
tivities (mg./ml.) were obtained when aliquots of 11-a (after ether removal) were incubated for 24 hr. under the conditions indicated: No additions or changes,0.8; plus 2.0 pg./ml. crotoxin, 0.8; plus 20.0 pg./ml. crotoxin, 1.5; plus 2.0 pg./ml. crotoxin and an ash of yeast extract equivalent to 20 mg./ml. of t,he yeast extract, 1.0; plus 2.0 pg./ml. crotoxin and a salt mixture,6 1.1; plus 2.0 pg./ml. crotoxin and dilution five times with 5 y0 sodium chloride, 1.8; and plus 20.0 pg./ml. crotoxin and dilution five times with 5 Y0sodium chloride, 2.2. In other experiments no effect was given by the addition of 0.002 M tricalcium citrate or 0.002 M calcium chloride. In contrast to the very low water solubility of lipovitellin and lipovitellenin, the crotoxin-treated lipoproteins readily dissolved in distilled water to give a 5 % solution. In the presenceof low and high concentrations of salt, however, their solubilities were more similar to those of the original proteins, i.e., insoluble in low (0.1-0.2 %) concentrations of salt but soluble in high concentrations. Although water solutions of crotoxintreated lipovitellin (1 X) were rapidly precipitated at 95-lOO”C., solutions in 5 $&salt did not precipitate after 10 min. Controls containing untreated protein in 5 Y0salt precipitated in less than 1 min. at 95-100°C. The phospholipides did not appear to be split from the lipoprotein complexes by crotoxin treatment. A serious problem in this interpretation was the very low solubility of the lysophospholipides in diethyl ether, which is empirically used as a solvent to remove “unbound” lipides from lipoproteins. However, partial precipitation from solution by salt or pH adjustment did not reduce the lysophospholipide content, and two attempts to differentially extract the lyso compounds with ethanol at low temperatures were unsuccessful. Ethanol extraction at room temperature removed the phospholipide, and highly active lysophospholipide preparations were obtained by ether precipitation of the ethanol extract. Hewdytic
Activities and Assays
Hemolysis was characterized by the low concentrations of lysophospholipides necessary and by the partial “all or none” or threshold requirements. The turbidity readings obtained in one experiment are plotted against the lysolecithin concentrations in Fig. 1. The typical “all 6 The salt mixture gave the following cations as the hydrochlorides in the concentrations (p.p.m.) indicated: K+, 10; Mg++, 2.5; Zn”, 0.25; Fe+++, 0.25; Mn++. 0.25; CO+++, 0.10; and Cu++, 0.10.
13;
-iCFFEC'TS OF CIEO'I'OXTN
or noLlc’~ or threshold characteristic is evident ill this ligure ill ,\vhich 1.-k ps./rnl. ga\-e only slight hemolysis while approximateI>, t\vic’r this cnolic*rntration (3.0 pg./ml.) gave estelisi\.e hcmolysis. The reproducibility of the qnantitati\e relationships was found to t)c depe&ent upon the blood sample employed, the method of dispersion of the lysophospholipide in preparation for assay, possibly the age of this dispersion, and unidentified factors. Nevertheless, a rather high 1
I
I
I
I
I
I 2
I 4
I 6
I 8
I IO
TIME FIG.
figures
IN MINUTES
AFTER
‘W
ADDITIONS
1. Hemolyses with graduated levels of lysophospholipide. Kumeric:J on curves refer to pg./ml. lysophospholipide (Prepn. 21-(1-12) added.
reproducibility was found in a series of 27 experiments with a total of 97 tests of the standard preparation 21-C-12. As compared on the basis of the percentages of tests at different concentrations requiring less than 20 min. for 70 % hemolysis, the following were the resulk obtained: 1.0-1.4 pg./ml., 0% (0 of 4); l-7-2.0 pg./ml., 25% (6 of 24); 2.3%2.i pg./ml., 76% (32 of 42); and 3.0-3.3 pg./ml., 89% (24 of 27). The preparation of samples was found partJic*ularly important. Apparently, the critical factor was the formation of a finely dispersed suspension or solution which was routinely accomplished by diluting ti ii-10 mg./ml. et,hanol solution lo- to 20-fold with water. This stock
138
FEENEY,
MACDONNELL
bND
FRAENKEL-CONRBT
suspension was usually diluted 3-6 times prior to assay, and 0.02-0.1 ml. was added per tube of 5 ml. of blood cell suspension. Although small amounts of ethanol were demonstrated to influence the hemolysis, the effects of the employed concentrations were small, and both standards and samples under assay were prepared similarly. A second way of preparation was solution by the addition of 0.1 N sodium hydroxide, with or without the addition of ethanol. The use of alkali, however, caused inactivation of some samples and was therefore not routinely employed. No direct hemolytic effect was exerted by the crotoxin in concentrations equivalent to those used in the hydrolyses, and it was therefore unnecessary to inactivate the enzyme prior to assay. The least accurate assays were obtained with whole yolk, crude yolk fractions, and the lipoproteins. Part of the difficulty with the lipoproteins was precipitation during the assay. This was a result of their unique solubilities; i.e., they were soluble in distilled water and strong salt but relatively insoluble in the dilute salt solution necessary for suspension of the blood cells. Although the possibility of precipitation made it necessary to assay the lipoproteins at mult,iple levels, it was usually possible to find a series of concentrations that gave slopes similar to the standard.
DISCUSSION Although phospholipides and their complexes are of major importance in the biophysical and biochemical systems of living materials, their actual roles are only very poorly understood. The present studies with crotoxin-treated lipoproteins and frozen and thawed egg yolk support the long-advanced hypothesis that the gelation of yolk on freezing and thawing is related to changes involving the lipoproteins (1, 7, 12). These results agree with the older studies of Tressler (13) in which gelation was reduced by treatment with the proteolytic enzyme pepsin or with crude enzymes. Other studies at this laboratory which will be reported elsewhere give still further data indicating the importance of the lipoproteins in this freezing-induced deterioration, as well as their roles in those properties of yolk which are responsible for heat coagulation. The importance of the phospholipides in the gross properties of yolk has also been indicated by studies with microorganisms. In such studies, precipitates were observed in media containing egg yolk and inoculated with members of the genus Clostridium (14) and certain Bacilli (15).
EFFECTS
OF
CROTOXIN
13!1
The Bacilli mere also shown to attack phospholipide preparations (15). However, only the lecithinase of t!he clostridia has been characterized, and this as lecithinase C (4, 14). Lecithinase C is a phosphatase and cleaves the phospholipide molecule bet’ween the phosphate and the glycerol components. The data suggested that crotoxin attacked the lipoprotein complexes directly without splitting the lipide from the protein, but conclusive proof of such action was not obtained. This was a result of the empirical criteria for defining a lipoprotein. The definition as usually employed defines that lipide, which is bound to protein to form a lipoprotein, is not extra,cted by the more nonpolar solvent,s such as petroleum ether or diethyl ether. The insolubility of the lysophospholipides in solvents of this nature makes it difficult to apply such an empirical formula. ,1 number of important problems still present themselves with regard to enzymatic action of crotoxin on the yolk constituents. Foremost among t,hese are the variable rates of reaction obt’ained and apparent lack of activity on partially purified cephalin. This activity of the enzyme on cephalin in unfract’ionated yolk and the absence of activit,y on the purified material confirm the observations of Chargaff and Cohen ilO), who post,ulated the possible presence of susceptible complexes of cephalin ill the intact yolk. Of interest in this regard are the many studies on t,he action of the lecit’hinase containing venoms of different snakes on erythrocytes (16). It is possible that the reason why certain venoms do not act upon erythrocytes concerns their capability of hydrolyzing cephalin. With regard to egg yolk it is important t.o determine whet,her or not t,here is an enzymatically susceptible complex of cephalin in egg yolk and consequently whether or not the isolated cephalin is an artifact. So at,tempt was made in these studies to refine the hemolytic assay developed. Of the various factors discussed in the text, the most important appeared to be the state of lysophospholipide in the dispersions employed for assay. It should be possible, by better standardization of sample preparations, to employ the hemolytic assay for preparations t,hat do not precipitate on dilution in water or salt. The fact that au “all or none” characteristic or threshold amount is necessary for hemolysis might indicate a reaction with, or absorption by, the erythrocyte or ils constituents prior to the occurrence of hnmolysis. Surface-tcnsioll rcdilctiorl ( 13 is also probably a factor.
140
FEENEY, MACDONNELL
AND
FRAENKEL-CONRAT
SUMMARY
The gelation of egg yolk caused by freezing was reduced by incubation with crotoxin (lecithinase A) before or after freezing. The lipoprotein fractions of yolk, lipovitellin and lipovitellenin, were attacked by crotoxin. The lysophospholipoproteins formed were hemolytic, had changed solubilities in water, and could be split by ethanol to yield impure lysophospholipide (desoleophospholipide) and the protein moiety. The lipoprotein complex did not appear split by action of the enzyme. The possible relationships between the changed properties of the lipoproteins and the modified gelation characteristics of frozen yolk were discussed. The hemolytic activities of yolk fractions treated with crotoxin were studied. With purified lysophospholipide preparations hemolysis of rat and rabbit erythrocytes exhibited a partial “all or none” characteristic. REFERENCES 1. URBAIS, O., AND MILLER, J., Znd. Eng. Chem. 22, 355 (1930). 2. SLOTTA, K. H., AND FRAENKEL-CONRAT, H. L., Ber. 71B21076(1938). 3. DELEZENNE,~., ANL)FOURNEAU,E., BulZ.soc.chim.[4] 16,421 (1914). 4. CELMER, W. D., AND CARTER, H. E., Phgsiol. Revs. 32, 167(1952). 5. PORGES, N., Science 117, 47 (1953). 6. ALDERTON, G., AND FEVOLD, H. L., Arch. Biochem. 8, 415(1945). 7. FEVOLD, H. L., AND LAUSTEN, A., Arch. Biochem. 11,1 (1946). 8. MECHAM, D. K., AND OLCOTT,H. S.,J. Am. Chem. Sot. 71,367O (1949). 9. OWENS, H., Food Znds. 19, 606 (1947). 10. CHARGAFF, E., AND COHEN, S. S., J. Biol. Chem. 129, 619 (1939). 11. LEVENE, I?. A., ROLF, I. P., AND SIMMS, H. S., J. Biol. Chem. 68,859 (1924). 12. LEA, C. H., AND HAWHE, J. C., Biochem. J. (London) 62,105 (1952). 13. TRESSLER, S. K., U. S. Patent 1,870,269 (1932). 14. MCCLUNG, L. S.,HEIDENREICH, P., AND TOABI, R., J. Bacterial. 61, 751 (1946). 15. COLMER, A. R., J. Bacterial. 66, 777 (1948). 16. ROY, A. C., Nature 166,696(1945). 17. PETHICA, B. A., AND SCHULMAN,J. H., Biochem. J. (London) 63,177 (1953).