Convenient apparatus for preparative polyacrylamide gel electrophoresis and its use in purification of three porcine neurophysins

ANALYTICAL

BIOCHEMISTRY

Convenient

44, 174-181

Apparatus

for

Electrophoresis

Preparative

and

of Three DAVID

(1971)

Its Use

Porcine

H. ,COY

Polyacrylamide

Gel

in Purification

Neurophysinsl

AND

TING-CHI

Department of Biochemistry, The 945 South Detroit Avenue,

ReceivedApril

Medical Toledo,

WUU College of Ohio, Ohio 43614

30, 1971

The neurophysins, present in the posterior pituitary lobes of several mammalian species, are proteins which bind the peptide hormones oxytocin and vasopressin. It has been suggested (1) that they may be involved in the mechanisms that control the storage and release of these hormones. Different components of neurophysin have been isolated from bovine (2,3) and porcine (4,5) posterior pituitary glands. The porcine neurophysins have been isolated from an acetone-dried powder of pig posterior pituitary lobes by two methods. In the first (4), the powder was percolated with a discontinuous gradient of water and acetic acid in ethanol to give a number of crude protein-containing fractions. Neurophysin I (Fig. ‘3) was obtained from these by a series of gel filtration and ion-exchange chromatography steps. Later, neurophysins II and III were also separated from the percolation fractions (6). In the second method (5), a protein-hormone complex was isolated from the powder by extraction with 0.1 M HCl and precipitation with sodium chloride. Pure samples of the three neurophysins were obtained from this by Sephadex and ion-exchange chromatography. The fact that the neurophysins exhibit well-separated bands following electrophoresis in starch gel (5) suggested that preparative-scale polyacrylamide gel electrophoresis might offer a very rapid technique for the final stage of purification of the proteins in relatively large amounts. For this purpose we designed a simple and readily constructed apparatus which consistently gave excellent separations of both standard test protein mixtures and the neurophysins. MATERIALS

AND METHODS

Apparatus. A schematic representation of the assembled system is shown in Fig. 1. The outer column is constructed from Pyrex tubing (3.5 ‘This

work

was supported

by

NIH

contract

174

69-2193.

SEPARATION

OF

PORCIiYE

175

NEVROPHYSlNS

(+

4

-K

-

I

-L

I--Fm. 1. Preparative electrophoresis apparatus: (A) outer glass column (3.1 cm i.d., 25 cm high) with side arm; (B) inner glass cooling tube (1.2 cm 0.d.); (C) elution rap made from base of polyethylene bottle (3.8 cm i.d.) and pierced at regular intervals with 8 slits (10 mm long, 1 mm high) ; (D) adjustable rubber gasket over which elution cap fits tightly: (E) narrow-bore elution tube made from polyethylene t,ubing PE 200; (F) rubber seal made from rubber stopper No. 00; (G) 7.5 cm layer of separator gel; (H) 2.0 cm layer of “stacking” gel; (I) level of cathodtl buffer; (J) lcvcl of anode buffer; (Ii) anode buffer replaced at same rate as elution flow; (I,) coolant in; (M) coolant out; (N) eluant to TTV monitor and fraction collector: (0) platinum wire cathode; (P) platinum wire anode ; (Q) rubber stopper No. 5; (R) jacketed, 1 liter containing vessel for anode buffer; (S) elution slits.

cm o.d., 3.1 cm i.d.) fitted with a V 24/i& female joint and a small side arm positioned at a distance of 17 cm from the base of the column. The inner cooling column consists of a ‘S 24/&, male joint equipped with a reduced diameter (1.2 cm o.d., 1.0 cm i.d.1 lower tube which is placed within the larger container so that the bases of both tubes are level. During electrophoresis, material travels down a vertical cylinder of acrylamide gel held by adhesion between the walls of the inner and outer tubes. As consecutive zones of separated substances emerge from the lower gel surface they enter a continuous and symmetrical stream of buffer which is drawn inward through the clution cap slits. The solution lea,ves the apparatus via narrow-bore plastic tubing threaded

176

COY

AND

WUU

through the central cooling tube and passes to an ultraviolet absorptionmeter (Isco model UA-2) and a fraction collector. The elution buffer also serves as the lower electrode (anode) medium and is replenished at the same rate at which it is removed. The upper cathode buffer, resting on top of the gel, may be renewed either manually at regular intervals during a fractionation or continually by means of a pump. For all the pumping operations required by the apparatus a Technicon proportioning pump (model 105-A200-01) with calibrated tubing was found to be ideal. Ohmic heating, which might cause distortion of both the gel and the migrating protein bands, was avoided by passing a suitable cooling liquid through the inner tube and the jacket of the anode vessel. Buffer Systems. Solutions chosen for fractionation of the neurophysins were based on the Tris-glycine system of Schenkein et al. (7). The cathode compartment buffer and the separator gel were made up to contain, per liter, 1.5 gm tris(hydroxymethyl)aminomethane (Tris) and 7.2 gm glycine at pH 8.4 (20°C). The anode vessel was filled with a solution containing twice the concentrations of Tris and glycine. The ionic strength of the lower buffer was increased to ensure that proteins emerging from the bottom of the gel encounter high conductivity and a low voltage gradient, thereby decreasing the tendency to diffuse out of the elution cap. It was then possible to use a slower elution flow rate for a given voltage, which results in less sample dilution. No apparent retardation of protein bands at the base of the gel was caused by passage of anode buffer into the matrix. Gel Composition: Prevention of Swelling. A separating gel containing 10% acrylamide and 0.25% N,N-methylenebisacrylamide (Bis) was found to give consistently the best elution pattern. There have been a number of reports (8-11) on the problems of band deformation caused by distortion and swelling of the anodal surface of the gel during electrophoresis. This has been attributed to excessive heat buildup within the core of the gel, but more recently it has been suggested (11) that ion accumulation at the interface, resulting in changes in the gel pore size, is the principal factor involved. In one system (II), this was overcome by using a low current setting during a run. However, this necessitates utilizing extremely low buffer concentrations so that a high enough voltage gradient may be created for a reasonably rapid fractionation time. We found during our investigations that the degree of gel distortion appeared to be largely dependent on the degree of cross-linking in the gel. The use of 0.25% Bis was found to prevent completely any visible signs of swelling after the passage of a 30 mA current for a total of 13 hr.

SEPARATION

OF

PORCINE

NEUROPHYSINS

177

At lower Bis concentrations, successively, greater degrees of distortion occurred. Furthermore, it seemed to be essential that gels with a lower acrylamide content should possess a correspondingly greater amount of cross-linking to survive the test conditions. For instance, a preparation 7.0% in acrylamidc required 0.36% Bis for good stability. Gel Preparation. Before mixing the gel solutions the elution aperture was blocked with a small square of adhesive tape. The lower end of the assembled column was covered with a sheet of Parafilm held tightly in place with a rubber O-ring. The whole apparatus was then clamped in a vertical position. Acrylamide (Eastman electrophoretic grade, 5.0 gmj, Bis (0.125 gmj, and N,N,N’,N’-tetramethylethylenediamine (TEMED, 30 ,uul) were dissolved in distilled water (15 ml). To this were added lower electrode buffer (25 ml) and a solution (10 ml) containing ammonium persulfate (0.15 gm/liter) . The mixture was carefully loaded into the column via the side arm and immediately layered with ca. 3 mm water, delivered from a dropping pipet to which was attached a short length of small-bore polyethylene tubing held close to the meniscus of the gel solution. At room temperature polymerization usually occurred within 30 min of adding the persulfate catalyst, resulting in a gel of length 7.5 cm and cross-sectional area 6.41 cm?. At least 1 hr was allowed for complete polymerization to take place. The base of the column and the elution aperture were carefully exposed and the elution cap was fitted and aligned to give uniform slit heights of 1 mm. The apparatus was then immersed up to the side arm in the anode buffer. The water layer on top of the gel was removed and the cathode compartment filled with buffer. Analytical Acrylamide Gel Electrophoresis. Electrophoresis on a small scale was carried out on a commercially available instrument (Canalco) using the same gel concentration and buffer systems as in the preparative-scale apparatus. Usually 10 pg of sample, dissolved in 10 pl of the upper buffer containing 5% sucrose, were layered on columns of gel (0.5 X 5.0 cm) and electrophoresed at approximately 5 mA/tube. Protein bands were visualized with amido black dye and destained electrophoretically. Preparation of Starting Material. A partially purified mixture of the three porcine neurophysins was obtained from acetone-dried pig posterior pituitary powder (Canada Packers Ltd., Toronto, through the courtesy of Ayerst Laboratories, Montreal) by the method developed by Hollenberg and Hope (3). The powder was extracted with 0.1 M HCl to yield a solution which was neutralized and centrifuged to remove a slight precipitate. A crude

178

COT

AND

WCU

neurophysin-hormone complex was isolated from the reacidified supernatant (pH 3.9) by precipitation with sodium chloride and further purified by elution in 0.2 M acetic acid down columns of Sephadex G-25 and G-75, respectively. The starch gel electrophoresis pattern of the extracted material has been reported (5), and is virtually identical with the one we found on analytical acrylamide gel electrophoresis (Fig. 3,M). Operation of the Preparative-Scale Column. A flow of water at ca. 10°C through the inner cooling tube and the anode vessel jacket was started immediately to prevent excessive heating in the gel. Before applying a protein sample the gel was pre-electrophoresed for about 2 hr at a constant current of 30 mA. The voltage at first rose and then fell, finally stabilizing at 320V. During this initial operation the elution flow was set at 1.2 ml/min to remove UV-absorbing material issuing from the gel. ‘The current was interrupted at the end of the cleaning period and the upper buffer completely replaced, leaving the system in readiness for the actual fractionation run. RESULTS

Fractionation of the Porcine Neur.ophysins. Samples of the protein mixture were dissolved in a minimum quantity of the upper buffer containing 50/o sucrose and one or two drops of 0.01% bromophenol blue tracking dye. The solution was then carefully layered on top of the gel surface; the elution flow was started at 0.8 ml/min, and the cooling flow at about 1 liter/min. A current of 10 mA was applied until the whole of the sample entered the gel matrix and then increased to 25 mA/250V for the duration of the run. Sharp refractile zones corresponding to the principal three components of the starting mixture were clearly visible moving down the gel. The eluant was read continuously at 253 nm and collected in 3.6 ml fractions. Sample Load and Layering-Solution Volume: Use of a “Stacking” Gel. It should be emphasized that narrow band formation is wholly dependent on the volume in which protein is layered on the gel surface. The maximum volume that did not result in impaired band resolution was in the order of 0.7 ml, in which a total of 30 mg of protein could be dissolved. For the fractionation of greater sample loads it was necessary to add a 2 cm layer of a concentrating gel to the already pre-electrophoresed lower gel. This layer was made according to the Tris-HCI (pH 6.9) formulation described by Davies (12). Excellent resolution could then be consistently obtained with up to 60 mg of sample dissolved in buffer volumes of up to 3 ml.

SEPARATION

OF

PORCINE

179

NEI-ROPHYSINS

Two representative elution patterns obtained with the two methods of sample application are shown in Fig. 2. Desalting of Fractionated Material: Yields. Fractions containing portions of the three major peaks were pooled and lyophilized. Tris and glycine were removed by chromatography on Sephadex G-25 in 0.2M acetic acid (column 2.2 x 110 cm). On desalting, tube numbers (Fig. 2) 14-19, 23-29, and 37-44, obtained from a 60 mg run, gave 15 mg, 9 mg, and 3 mg of purified material, respectively. Electrophoretic Homogeneity of the Isolated Components. The analytical acrylamide gel patterns shown in Fig. 3 illustrate that neurophysins I, II, and III were isolated in high purity even when large parts of their individual elution peaks were taken. Homogeneity was also demonstrated by electrophoresis in slabs of 15% starch gel, carried out by the method described by Smithies (13) with the buffer systems (pH 8.1) of Ferguson and Wallace (14). Homogen.eity of Components bly -4rnino Acid ilnaLysis and Digestion 0.6 i 0.5 -

A-

B “......... 0.4 0.3 .

0

:‘\

IO

20

TUBE

30

40

50

60

l

NUMBER

Fro. 2. Preparative ncrylsmide gel electrophoretic elution patterns for purified mixture of porcine neurophysins: (A) 60 mg of the mixture was dissolved in 2 ml cathode buffer, containing 5% sucrose, and layered on the surface of a 2 cm polyacrylamide “stacking” gel. This, in turn, rested on a 7.5 cm, 10% acrylamide separator gel. A 10 mA current was applied unt,il all the sample had entered the gel matrix and then increased to 20 mA/250V for duration of t,he run. Gel temperature was maintained at ca. 10°C and elution flow rate at 0.8 ml/min. 3.6 ml fractions were collected. (B) 30 mg of the same mixture was electrophoresed under identical conditions. However, the sample. dissolved in 0.7 ml buffer. was layered directly on top a 7.5 cm separator gel. The first tube number in the graph is the one in which tracking dye first appeared. Solid bars indicaie fractions of each peak which were poolrtl.

180

COY

AND

WUU

FIG. 3. Analytical acrylamide gel electrophoresis of purified components. Migration was toward top of photograph (to the anode). Protein zones were stained with amido black dye. Samples which exhibited patterns I, II, and III were obtained from fractions 14-19, 23-29, and 37-44, respectively. Pattern M is that of the neurophysin mixture which was fractionated.

with Trypsin.

Amino acid analyses were conducted on samples of each of the three purified proteins after their hydrolysis in 6 N HCl (22 hr). In each case the data agreed closely with those obtained from the analysis of samples of the neurophysins derived from other purification techniques. Also, when tryptic digests of the oxidized proteins were “fingerprinted” (electrophoresis on paper followed by chromatography in a perpendicular direction), patterns developed with ninhydrin were identical with those exhibited by standard samples. DISCUSSION

A major difference between the apparatus described here and many of those reported in the literature, several of which are commercially available, lies in the use of the anode buffer as the elution medium. This has enabled to be rapidly’and cheaply constructed an apparatus of extremely simple design in which efficient cooling of both the inner and outer gel surfaces may be accomplished. The buffer system used was found to be highly effective in the separation of the neurophysins. However, several other buffer systems have been tried and were compatible with the design. From our investigations it would appear that the serious problems many workers have experienced with distortion of the anodal surface of the gel may be readily overcome by choosing a suitable degree of crosslinking for a particular acrylamide formulation. In this way severe limitations to the size of current used for electrophoresis need no longer be applied. Due to the limited solubility of the protein preparation, it was neces-

SEPARATION

OF

PORCINE

181

NFXJROPHYSINS

sary to limit the weight of material to be applied directly to the separator gel. When an intermediate layer of a “stacking” gel was employed the weight could be doubled with no loss of resolution. However, an inherent disadvantage to using the additional concentrating layer is that the whole column of gel may be used only once, whereas a single separator gel could be reused for at least four runs with no loss of resolving capacity and a great saving in time. ADDENDUM

The use of a lower electrode buffer as the elution medium in the analytical-scale separation of RNA mixtures by electrophoresis on agarose gels has recently been reported by Popescu et al. [Anal. Biochem., 40, 247 (1971) ] . SUMMARY

A simplified and effective preparative polyacrylamide gel electrophoresis apparatus is described. It has been used to isolate rapidly three porcine neurophysins in sufficient quantities for sequencing studies that are at present in progress. ACKNOWLEDGMENTS

would like to thank Dr. M. Saffran for his generous support Crumm for valuable assistance rendered during this work. We

and Mrs. Sara

REFERENCES 1. FAWCETT, C. P., POWELL, A. E., AND SACHS, H., Endocrinology 83, 1299 2. BRESWW, E., AND ABRASH, L., Proc. Nat. Acad. Sci. U. S. 58, 640 (1966). 3. HOLLENBERCI, M. D., AND HOPE, D. B., Biochem. J. lQ6, 557 (1968). 4. Wuu, T. C., AND SAFFEAN, M., J. Biol. Chem. 244, 482 (1969). 5. UTTENTHAL, L. O., AND HOPE, D. B., Biochem. J. 116, 889 (1970).

6. WUU, T. C., unpublished results. 7. SCHENKEIN, I., LEVY, M., AND PEDDRICK,

(1968).

W., Anal. Biochem. 25, 387 (1966). 8. HJERT~N, S., JERSTEDT, S., AND TISELIUS, A., Anal. Biochem. 27, 108 (1969). 9. JOVIN, T., CHRAMBACH, A., AND NAUGHTON, M. A., Anal. Biochem. 9, 351 (1964). 10. GORDON, A. H., AND LOUIS, L. N., Anal. Biochem. 21, 190 (1967). 11. KAWATA, H., CHASE, M. W., ELYJIW, R., AND MACHEK, E., Anal. Biochem. 39, 93 (1971). 12. DAVIES, B. J., Ann. N. Y. Acad. Sci. 121, 211 (1965). 13. SMITHIES, O., Biochem. J. 61, 629 (1955). 14. FERGUSON, K. A., AND WALLACE, A. L. C., Nature 190, 629 (1961).