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Biochimica et Biophysics Acta, 380(1975)35’7-505. 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA Report BBA 51179

A SIMPLE AND HIGH YIELD PURIFICATION OF CROTALUS

ADAMANT&W3 PHOSPHOLIPASES AZ

MICHAEL A. WELLS ~e~rt~ent of geochemistry, 85724 (U.S.A.) (Received November llth,

College of ~e~ieine,

University of Arizona, Tucson, Ark

1974)

Summary A new, high yield, procedure for the purification of the phospholipases AZ of Crotulus adamanteus venom is described. The procedure involves precipitation of the bulk of venom proteins with 50% isopropanol, precipitation of the enzymes from the isopropanol soluble material with neodynium chloride, and final p~ifi~tion on DEAE cellulose. An overall yield of approximately 80% is achieved.

Wells and Hanahan [l] described a procedure for the purification of phospholipase A2 from Crotalus adamanteus venom which yields two highly pure and crystalline proteins termed phospholipases A2 -cyand -0. Although this procedure has been used for several years in this laboratory, it has some drawbacks. In particular the rather low yield (35%) and numerous chromatographic steps make preparation of large amounts of pure enzyme somewhat unattractive. A more thorough understanding of the properties of these enzymes has led to a new purification procedure which is described in this communication. Enzyme activity was measured by either of the following procedures: egg yolk phosphatidylcholines in diethyletherlmethanol [l] ; monomeric dihexanoyl phosphatidylcholine using 5 mM substrate, 0.1 mM CaClz , pH 8.0 and 30” C [ 21, micellar dioctanoyl phosphatidylcholine using 5 mM substrate, 0.1 mM CaClz 0.01 M MgClz , pH 8.0 and 40°C [3]. Substrates were prepared as previously described [ 1,3,4] . Protein concentration was determined from the absorbnace at 280 nm assuming Ai% = 10.0 [Il. DEAE cellulose was Whatman DE-52 (Reeve Angel, Clifton, N.J.). Neodynium chloride was obtained from Ventron Corp. (Beverly, Mass.). Other chemicals were reagent grade. Crotulus adamanteus venom was purchased from the Miami Serpentarium (Miami, Fla.).

5 g of venom are dissolved in 250 ml of 0.05 M acetate buffer pH 5.25 containing 10 mM CaCIZ . To this solution is added dropwise, over 30 min and with rapid mixing at room temperature, 250 ml of isopropanol. The suspension is stirred at room temperature for 30 minutes and then centrifuged in a Sorvall RC-BB, using a GSA head, at 5000 rev./min for 30 min at 4°C. The slightly turbid supernatant solution is decanted and the precipitate discarded. To the supernatant solution is added 10 ml of 0.5 M NdC& in 0.05 M acetate buffer pH 5.25. The suspension is mixed at room temperature for 30 min, and centriguged as described above. The supernatant solution is discarded and the precipitate dissolved in approximately 50 ml of 0.05 M Tris. HCI buffer, pH 8.0, confining 0.01 M EDTA. The solution is dialyzed in the cold for 48 h against 4 liters of the Tris EDTA buffer. The dialysis buffer is changed twice at 18 h intervals. After dialysis the protein solution is centrifuged as described above and the supernatant solution concentrated to approx. 10 ml using an Amicon Diaflo apparatus and a UM 10 membrane. This protein solution is applied to a 1.8 X 100 cm column of DBAE-cellulose which had been packed and equilibrated in 0.05 M Tris. HCl, pH 8.0, containing 1 mM EDTA. The column is eluted at room temperature with an exponential gradient formed by using three chambers of a Buchler Varigrad. The first two chambers contained 400 ml each of 0.05 M TrisHCl, 1 mM EDTA, 0.02 M NaGI, pH 8.0 and the third chamber an equal weight of 0.05 M Tris HCI, 1 mM EDTA, 0.2 M NaCl, pH 8.0. A flow rate of 50 ml/h was maintained with a peristaltic pump, and the entire gradient was collected in 5 ml fractions. Disc gel electrophoresis was carried out as previously described [l] and in some cases the protein was crystallized [l] .

TABLE I SUMMARY OF THE PURIFICATION OF PHOSPHOLIPASE A, FROM CROTALVS VENOM. IN EACH CASE A 5 g BATCH OF VENOM WAS USED, step

1.

Dissolved VtXiORi

2.

1sopropanol Solubfe

3.

NdCI, precipitate after dialysis and centrifugation

4.

DEAE-Cellulose fractions

o!

P ___I_ *DEAE

% protein

% activity

Purification

100

100

-

22.6 23.1 26.2 24.8

94.7 97.1 99.6 98.2

4.2 4.2 3.8 4.0

7.2 * 6.9 6.8 ** 6.9

85.8 87.1 90.6 88.3

11.9 12.6 13.3 12.8

1.98 2.05 2.10 2.00

48.1 50.3 52.0 51.0

24.3 24.5 24.8 25.5

1.28 1.25 1.20 1.20

30.0 30.5 30.8 29.5

23.4 24.4 25.6 24.6

column shown in Fig. 1B. ** DEAE column shown in Fig. 1A.

ADAMANTEUS

503

The results of four separate experiments are summarized in Table I. Three different assay procedures were used and the extent of purification was independent of the assay procedure used. For comparison to the earlier procedure [l] , the specific activity of the purified material was 1450-1500 using phosphatidylcholine in ether/methanol. Crystallization of the enzyme did not increase the specific activity. Disc gel electrophoresis of 100 pg of the enzymes isolated by ion exchange chromatography showed a single band. The patterns were identical to those reported previously [l].The enzymes isolated by this procedure are identical in all respects to enzymes isolated by the previous method; including amino acid analysis [ 11, molecular weight [1] , solvent induced spectral perturbations [ 51, cation induced spectral perturbations [6], kinetic properties [3,4], and chemical modifications [7]. The obvious advantage of the current procedure is the high yield of enzyme, approx. 80%, and the relative ease of the purification scheme. Successful application of the procedure depends on careful attention to details in each step. The more critical aspects are as follows: (1) Successful removal of the bulk of the protein by alcohol precipitation depends on the pH of the solution, the presence of CaCl, , and the alcohol TABLE II EFFICIENCY

OF VARIOUS

ALCOHOLS

IN PRECIPITATING

PROTEINS

In each case the crude venom was dissolved in 0.05 M acetate buffer PH 5.25 containing 10 mM C&l,. The protein was precipitated by addition of an equal volume of alcohol. The results refer to the amount of protein and enzyme activity remaining in the supernatant solution after centrifugation, Alcohol

% protein soluble

% activity soluble

Methanol Ethanol n-Propanol Isopropanol

38.7 37.9 30.9 21.6

98.1 97.3 98.0 98.9

TABLE III EFFECT OF BUFFER pH AND CaCl, CONCENTRATION ON THE RECOVERY ENZYME ACTIVITY DURING PURIFICATION OF PHOSPHOLIPASE A,

OF PROTEIN

AND

Alcohol fractionation was carried out using isopropanol. Buffers were prepared using 0.05 M acetate and the indicated concentration of CaCl, . Conditions

% protein soluble

96 activity soluble

(A) Recovery of protein and enzyme activity after alcohol fractionation pH 5.00. 0.01 M CaCl, pH 5.25, 0.01 M CaCl, pH 5.50, 0.01 M CaC1,

28.4 22.4 35.9

98.3 98.6 99.0

PH 5.25, no CaCl, PH 5.25. 0.01 M CaCl, pH 5.25, 0.1 M CaCl,

30.0 22.4 60.1

98.1 98.6 97.4

Conditions

% protein

% activity

(B) Recovery of protein and enzyme activity after alcohol fractionation and subsequent NdCl, tation (measured after dissolving the precipitate and centrifugation pH 5.00, 0.01 M CaCl, PH 5.25, 0.01 M CaCl, pH 5.50. 0.01 M CaCI,

9.0 7.0 12.6

83.0 80.7 80.6

pH 5.25, no CaCl, pH 5.25, 0.01 M CaCl, pH 5.25, 0.10 M CaC1,

5.5 7.0 14.4

50.3 88.7 87.1

precivi-

504

used. As shown in Table II, isopropanol is most effective in the alcohol precipitation step. Table III shows that a pH of 5.25 and a CaC& concentration of 0.01 M are required for maximal recovery of enzyme activity and also yield the highest purification after NdC& precipitation. (2) A detailed study of precipitating ions was not carried out. It was, however, noted that the divalent ions Ca2’, Ba”, Znzt and Cu2+ gave very little precipitation of the enzyme from the isopropanol soluble fractions. NdC13 was used because it is readily available and gives stable solutions. (3) The gradient used to elute the enzyme from DEAE cellulose was changed from the linear gradient used previously. This was necessary in order to separate proteins which eluted close to the e-enzyme. There was some variation in the elution profiles depending on the batch of venom used. Two extreme examples are illustrated in Fig. 1. In each case electrophoretieally

FRACTION

NUMBER

Fig,l. DEAE-cellulose fractionation of phospholipase A,. Two different samples were fractionated on a 1.8 X 100 cm column using an exponential gradient. The flow rate was 50 mi/h and 5-ml fractions were collected. a and prefer to the two forms of the enzyme.

pure enzyme was obtained. This new gradient seems capable of handling the variation in the protein composition of the solution applied to the DEAE column. The pattern illustrated in Fig. 1A was obtained in three of four cases described in Table I, whereas the pattern obtained in Fig. IB was obtained only once. No explanation is available to explain these differences. (4) It should be noted that relatively high EDTA concentrations are required to redissolve the protein after precipitation with NdC13 . Based on the kinetic and spectral properties of the purified enzymes, it is concluded that no Nd3” contaminates these preparations. The high yield and simplicity of this ~ur~ication scheme partially compensates for the relatively high cost of snake venom, and should allow pure phospholipase A2 to become more readily available. Mrs B. Geest provided expert technical assistance. This work was supported by a grant from the National Science Foundation (BMS 74-19351).

505

References 1 2 3 4 5 6 7

Wells, M.A. and Hanahan. D.J. (1969) Biochemistry 8, 414-424 Wells, M.A. (1972) Biochemistry 11,1030-1041 Wells. M.A. (1974) Biochemistry 13. 2248-2257 Misiorowski, R.L. and Wells, M.A. (1973) Biochemistry 12, 967-975 Wells, M.A. (1971) Biochemistry 10, 4078-4083 Wells, M.A. (1973) Biochemistry 12,1080-1085 Wells. M.A. (1973) Biochemistry 12,1086-1092

A simple and high yield purification of Crotalus adamanteus phospholipases A2.

501 Biochimica et Biophysics Acta, 380(1975)35’7-505. 0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA Report BB...
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