Electrofocusing and Gel Electrophoresis of Bovine Neurophysins

Transcription

1 Eur. J. Biochem. 28, (1972) Electrofocusing and Gel Electrophoresis of Bovine Neurophysins Analytical and Preparative Experiments Vladimir PLSEA, Jeffrey F. MCKELVY, and Howard SACHS Roche nstitute of Molecular Biology, Nutley, New Jersey (Received December 9/March 15, 1972) The isoelectric points of bovine neurophysins and 1 were estimated by isoelectric focusing in a sucrose gradient and in polyacrylamide gels. The values found were 4.43 and 4.97 for neurophysins and 11, respectively, and were in agreement with the observed relative electrophoretic mobilities in disc gel electrophoresis. Electrofocusing in gels resolves the components of crude neurophysin better than disc electrophoresis. Neurophysins and 1 obtained by electrofocusing in a sucrose gradient were about as pure as preparations obtained by ionexchange chromatography. The neurophysins are a group of low molecular weight proteins found in the posterior pituitary glands of several vertebrate species. These proteins form noncovalent complexes with the neurohypophysial hormones and are presumed to play a role in the storage and release of the hormones in vivo. At least three neurophysins, designated, 1 and C, have been identified in acidic extracts of acetonedried bovine pituitary tissue [l, 21. The neurophysins exhibit different electrophoretic mobilities and differences in amino acid composition, so that differences in their isoelectric points may therefore be anticipated. n this communication we report the isoelectric points of neurophysins and 1 as estimated by the electrofocusing technique (in a sucrose gradient or in polyacrylamide gel) and discuss the possibilities which this technique offers for the identification of neurophysins and 1 in tissues and for their separation from tissue extracts and for the purity control of neurophysin preparations. The results are compared with those obtained by gel electrophoresis. EXPERMENTAL PROCEDURE Preparatiofi of Neurophysins Acetone powder was either prepared from commercial frozen bovine posterior pituitaries (PelFreez Biologicals, nc., Rogers, Arkansas) or obtained from ParkeDavis Company (Detroit, Michigan). The extraction and fractionations by ph and NaCl precipitation (10 g NaCl per 100 ml) were carried out according to Hollenberg and Hope [3]. Subsequent Abbreviation. soelectric point, P. steps were as follows : The NaCl precipitate obtained from 40 g of acetone powder was dissolved in 250 ml water with the addition of several drops of concentrated ammonium hydroxide ; the clear solution was reduced in volume to about 100 ml by ultrafiltration through a Diaflo UM 05 membrane under nitrogen pressure (3.5 atm), and washed with approximately 500ml wasr. The retained protein solution was acidified (1 ml glacial acetic acid per 9 ml solution) and the small proteins were washed through a Diaflo PM30 membrane with 1000 ml of loo/, acetic acid (nitrogen pressure 1.4 atm). The ultrafiltrate was concentrated in a rotary evaporator at 30 C, and lyophilized. The lyophilized material ( g) was dissolved in approximately 10 ml of 1 M acetic acid and passed through a column of Sephadex G25 (2.5 x 100 cm) equilibrated in 1 M acetic acid, to yield g of hormonefree proteins (Fig. 1). The chromatographic separation of neurophysins and 1 was performed on DEAESephadex A50 using either a discontinuous ph gradient of (pyridine buffer, approximately 0.85 M) [4,5] or an NaCl gradient [2] of 00.3 M. Fractions containing neurophysins and those containing neurophysins 1 (identified by polyacrylamide gel electrophoresis, see below) were separately pooled, and the salt was removed by ultrafiltration through a Diaflo UM05 membrane; the pyridine buffer was removed by repeated evaporation and the sample was lyophilized. Some samples used in our experiments were prepared by the previously described procedure [5] and kindly placed at our disposal by Dr Esther Breslow (Department of Biochemistry, Cornell University Medical School, New York).

2 V01.28, No. 1, 1072 V. LBRA, J. F. MCKELVY, and H. SACHS oo 22.. parts. One of them was fixed in 12O/, trichloroacetic acid for 6 h and the intensity of protein precipitate in the bands wits measured with a JoyceLoebl UV Polyfrac at 260nm. The second one was sectioned with a horizontal gel slicer into 2.5mm slices which were each extracted overnight with 1 ml water for ph determinations (glass microelectrode, Radiometer, Copenhagen) Vel Fig. 1. Dissociation of neurophysin. peptide complex (van Dyke protein) on Sephadex G25 in 1 M acetic acid. Column: 2.5~90 cm, flow rate 0.5 ml/min. V,1 is relative elution volume, taken as the ratio of effluent volume to void volume. Numbers at arrows indicate the relative elution volumes ( Vel) for a particular peak. Protein peak, Vel = 1.12 to 1.24, oxytocin, Vel = 2.00, arginine vasopressin, Vel = 2.15 (peptides identified by thinlayer chromatography 1141 and by bioassay) Preparative PolyacrylamideGel Electrophoresis The preparative polyacrylamide gel electrophoresis of neurophysins or 1 separated from a crude preparation by either of the chromatographic procedures described above was carried out on a 150/, polyacrylamide gel column (length 7580 mm, volume ml) at ph 9.5 (Trisglycine buffer, containing 3.0 g Tris and 14.4 g glycine in 1 1) using a Canalco PrepDisc apparatus. The samples, 5 15mg, were applied in a 50 /, sucrose solution containing electrophoresis buffer and tracking dye (bromophenol blue) and the electrophoresis was carried out at 500V (516mA/cm2). The flow rate of buffer through the anode compartment was 0.4 ml/min ; 15min fractions were collected. The absorbance was monitored at 280nm using an LKB Uvicord recorder. Fractions constituting ultravioletabsorbing peaks were pooled and the buffer components were removed by ultrafiltration (UM 05) ; protein concentrations were estimated by the Folin reaction [ti]. The resulting solutions were examined for electrophoretic purity by analytical disc electrophoresis. The solutions containing electrophoretically pure components were lyophilized. Electrofocusing in Polyacrylamide Gels The gels (length 14 cm) were prepared according to Catsimpoolas [7] and Wrigley [S], using LKB ampholytes of ph range 36 (batch 8142) or 46 (batch 8132). The amount of sample applied per tube was pg. The anode solution was 4O/, H,PO,, the cathode solution 4,lo diethanolamine ; the electric field was applied for 5 h (200350V, 12mA per tube). Thereafter the gels were extruded and sliced with a longitudinal gel slicer (Canalco) into two equal Electrofocusing in a Sucrose Gradient Electrofocusing was carried out using the LKU Ampholine system. The 110 ml column (LKB 8101) was used with a linear density gradient, 0400/, sucrose, containing 1 2O/, ampholyte, ph range 46 (batch 8152) or 36 (batch 8142). The protein (2045 mg) was dissolved in 4.6 ml sucrose solution in the middle of the gradient. A potential of 500V was applied for 72 h (current after temperature equilibration ma) ; the column contents were then pumped out from the anode end. Fractions of 2 ml were collected and the absorbance at 275 nm was monitored with an SCO UV recorder ; the ph of each fraction was measured on a Radiometer model M26 ph meter. The separation of proteins from gradient components was achieved by chromatography on Sephadex G25 (medium, column size 2 x 58 cm), in 1 M acetic acid. Samples of 2 to 4 ml were applied and the column was eluted at a rate of 0.4 ml/min, 5ml fractions being collected ; absorbance at 280 nm was monitored with an LKB Uvicord 1 recorder. Analytical Disc Electrophoresis Lyophilized protein samples (4060 pg) were dissolved in 100 p1 of electrophoresis buffer (or loo$ samples of peak fractions were used) and mixed with an equal volume of 50 /, sucrose in electrophoresis buffer containing tracking dye (bromophenol blue). The samples were subjected to electrophoresis 7.5 or 15O/, gels, at ph 9.5 and 4.3, using procedures specified by Canal ndustries, nc. [9]. Following electrophoresis, the gels were fixed for 5 h in 12O/, trichloroacetic acid. Samples containing ampholyte were washed in 120/, trichloroacetic acid for 2 days with 5 changes of washing solution [7]. The gels were stained with freshly prepared Coomassie blue (2 h) and destained in loo/, acetic acid. Alternatively, the gels, after acid fixation, were examined on a Joyce Loebl UV Polyfrac as described above. RESULTS Gel Electrofocusing n a ph gradient from 4 to 6, crude neurophysin prepared by ionexchange chromatography can be separated into 8 components (Fig.2) with p values from about 4.2 to 5.4. We found this profile to be

3 ~~ ~ 112 Bovine Neurophysim Eur. J. Biochem. Os6 r PH Fig.2. Distribution profile of crude bovine neurophysin (170pg) after Ampholine electrofocuaing in polyacrylamide gel. The ph given is that of slices after extraction. Numbers at arrows indicate extrapolated ph values of peak tops E ci a, L) c $0.4 u) n Q EO.4 N " D r 0 R Q , Relative mobility Fig. 4. Diatribution profile of crude bovine neurophyain (60pg) after disc electrophoresk in 7.501, polyacrytamide gel at ph 9.5. TDtracking dye Table 1. soelectric points and intrinsic dissociation Constants (pkint) of bovine neurophyains Results are given as mean & standard error of mean; in parentheses, the number of measurements. EF = electrofocusing. Titrations were carried out at 25 "C, 0.16 ionic strength (see Breslow r51) Value Method Neurophysin p gel EF 4.45 f 0.06 (7) 5.01 & 0.04 (9) EF in sucrose gradient 4.42 f 0.17 (3) 4.92 f 0.08 (7) Weighted average Titration pkint Titration EF The value based on amino acid sequence of neurophysin 11 [Z; an earlier value (4.44) published by Breslow [51 was based on an older amino acid analysis. 1 Fig.3. Distribution profge of neurophysin (210pg) and neurophyain 11 (270 pg) prepared according to Breslow (51 after Ampholine electrofocusing in polyacrylamide gels. For symbols, see Fig.2 reproducible for different preparations of bovine acetonedried neurohypophysial tissue. Two major bands having p values of approximately 4.5 and 5.0 and corresponding to neurophysin and neurophysin 11, accounted for 2901, and 2701, of the total absorbance, respectively (determined planimetrically ). The p values of neurophysins and 1 were determined on highly purified preparations of both proteins. All such preparations obtained by ionexchange chromatography on DEAESephadex by either of the methods described above contained a small number of minor components in addition to the major band as shown in Fig.3. The content of these minor components accounted for less than loo/, of the total protein except for one preparation of neurophysin (purified on DEAESephadex in pyridine buffer [5]) where we found more than 25'1,. The pl values found for neurophysins and 1 are summarized in Table 1. We also found that the ph gradient in gels was reproducible. When we studied the dependence of p for highly purified neurophysin on the time of electrofocusing, we obtained constant values after one hour and for a time period of up to 12 h. Analytical Disc Electrophoresis The electrophoretic profile of crude neurophysin on a 7.5O/, gel at ph 9.5 is shown in Fig.4. From four to six bands are observed in different neurophysin preparations. The neurophysin band contains approximately 2201, of the total protein by absorbance, and the neurophysin1 band 35O/,. With

4 ~ ~~ ~ Vol.28, No. 1,1972 V. PLSKA, J. F. MCKELVY, and H. SAOHS 113 Table 2. Relative electrophoretic mobilities of bovine neurophysins on polyacrylarizide gels at ph 9.5 Tracking dye was bromophenol blue. Results are given as mean & standard error of mean; in parentheses, the number of measurements Method of Relative mobility of neurophysin gel electrophoresis 1 _ ~ Analytical, 7.501, gels 0.92 ho.01 (4) (4) Analytical, 15O/, gels 0.74 f 0.03 (6) 0.42 & 0.04 (6) Preparative, 150/, gels 0.67 f 0.08 (4) 0.34 f 0.02 (5) decreasing ph the mobility of all species present is decreased. At ph 4.3 (7.50/, gels) we observed little electrophoretic mobility, and at ph 2.3 slight cathodic mobility with very poor separation of bands. Relative mobilities (tracking dye : bromophenol blue) estimated as the ratio Dprotein/D,jye (D = the distance of the band from the top of the separation gel) at ph 9.5, using both pure and crude preparations, are summarized in Table 2. Electrofocusing of Crude Neurophysin in a Sucrose Gradient We attempted to use electrofocusing in a sucrose gradient on a preparative scale to purify neurophysins and 11. We observed some differences in the degree of resolution obtained from one experiment to another. Occasionally, minor peaks, in addit,ion to those shown in Fig.5, were observed. n most cases each peak comprises more than one species, as estimated by analytical disc electrophoresis and gel electrofocusing. The degree of purity of neurophysin obtained by electrofocusing in a sucrose gradient was considerably greater than that of neurophysin 11. The ph values measured at the maximum 280nm absorbance of each peak were taken to be the isoelectric points of the predominant species in the peak. These p values are summarized in Table 1. The neurophysin obtained from the electrofocusing column after a 72h run in most cases had the same purity as that prepared by ionexchange chromatography, i.e. it contained minor components with P values lower than that of neurophysin. Some preparations also contained a minor component having a P of approximately 4.7. Neurophysin 1 always contained a component with p lower than that but very close to that of neurophysin and very often also another component of (P The purity of any given preparation was independent of the choice of ampholyte and duration of electrofocusing, but seemed to be influenced by the rate at which the medium was drained from the column at the completion of the electrofocusing process. The components of crude neurophysin are very insoluble at their p values and appear as distinct white bands after several hours of electrofocusing. 8 Eur. J. Biochem., Vol ; =l P* Fig. 5. Distribution profile of crude bovine neurophysins (41.5 mg) after Ampholine electrofocusing in sucrose gradient. Electrolyte: lo/, ampholyte, ph 46. Numbers at arrows indicate interpolated ph values of peak tops E % A A vel Fig.6. Separation of neurophysin (4 rng, peak A) from ampholyte (peak c) and suc~ose (peak D) on Sephadex 625 (2 x 58.6 cm column) in 1 M acetic acid. Peak B is unidentified (observed also by Brown and Green [lo]). For symbols, see Fig. 1 We found that gel filtration on Sephadex G25 in 1 M acetic acid effectively separated the protein from the ampholyte and sucrose, as shown in Fig.6, though we did not have any definite evidence that the protein fraction was entirely free of ampholyte [lo, 111. When we attempted such a separation using Diaflo membrane filtration, we found that ampholyte would not pass through a UM2 membrane, even though the exclusion limits of the membrane indicated that it should. The yields were variable, depending on the degrees of resolution. When the fractions composing the centre of each peak were pooled, 7 to 1301, of total proteins were recovered as neurophysin 11. Preparative Gel Electrophoresis Using preparative polyacrylamide gel electrophoresis, we attempted to purify further the neurophysins and 1 prepared either by ionexchange chromatography or by electrofocusing in a sucrose gradient. The relative mobilities, estimated as a C

5 114 Bovine Neurophysins Eur. J. Biochem. ratio of the time in which a protein peak leaves the column to the corresponding time for the tracking dye (bromophenol blue) are given in Table 2. The elution profile was not entirely constant, but we usually observed small peaks in front of both neurophysins and 11. This fasterrunning component associated with neurophysin was indistinguishable from neurophysin by analytical disc electrophoresis, while, in the case of neurophysin1, the faster component was detectable by analytical disc electrophoresis. Whether these impurities were contained as minor components in the starting material and were unidentified by analytical disc electrophoresis because of a great disproportion in content and sma.11 differences in mobilities from the major component, or whether they were formed during electrophoresis in alkaline solution, has not been determined. Only the fractions from the center of each peak were pooled. These contained only about 30 /, of the original protein; the total protein recovery in all fractions as estimated by the Folin method [6] was 75 to 85O/,. The gel electrofocusing profile of pooled peak fractions from preparative gel electrophoresis was the same for neurophysin as before preparative electrophoresis whereas, in the case of neurophysin 11, the peak with p 5.4 was absent. DSCUSSON n the following we shall discuss three points, which might be of more general interest: (a) the analytical use of gel electrofocusing, (b) the possibilities for purification offered by column electrofocusing and preparative gel electrophoresis, and (c) estimates of the p l values of neurophysins and 11. We found analytical gel electrofocusing to give a protein profile of greater resolution, and with sharper bands, than either analytical or preparative polyacrylamide gel electrophoresis (Fig. 2 and 4). Electrofocusing thus should be a useful tool for studying protein profiles in neurohypophysial tissue extracts, e.g. under different physiological conditions, and presumably also for comparing neurohypophysial proteins from different species. At present it also seems to be the best routine technique for assessing purity during neurophysin purification. t turned out that even electrophoretically pure preparations of neurophysin or neurophysin 1 obtained by ionexchange chromatography as described contain minor components detectable by gel electrofocusing. Some of these components appeared in all preparations purified in either of the ways described. Because the p values of these minor components were always lower than that of the major one, it seems likely that they may be products of partial deamidation of the neurophysins. Gel electrofocusing may prove useful in kinetic studies of such processes. On the preparative scale, we were not entirely successful in the direct separation of pure components using electrofocusing in a sucrose gradient. As mentioned in the Results section, the purity of the products was strongly dependent on different circumstances, particularly on the rate with which the electrofocusing column was drained. One can imagine that protein species possessing similar p values, and therefore located in two close bands, will be partially mixed during the flow through the broad upper part of the column and at the bottom where some turbulent flow may be expected. n the upper broad part the turbulence is presumably much less compared to that at the bottom but can, nevertheless, influence the final separation of components initially located close to the top of the column, which have to travel a long distance to the bottom before complete elution. This may explain why preparations of neurophysin 1 appear less pure than those of neurophysin and often contain a component with a variable pl even through in many cases (when more than 2030 mg of material were used) a good separation could be observed before the column was drained. The yields of both neurophysins were somewhat lower than in case of ionexchange chromatography. Breslow [5] published values of 20 and 35O/, for the recovery of neurophysins and 11, respectively, and Rauch et al. [2] found similar values; our average yields obtained by both methods [2, 51 were 14 O/, for neurophysin and 36O/, for neurophysin 11. t must be stressed, however, that in our experiments the yield was a secondary consideration, our aim being the preparation of very pure material, with a better gelelectrofocusing pattern than the material from ionexchange columns. The same considerations apply to our use of preparative gel electrophoresis. The large number of electrofocusing experiments carried out allows an estimate of pl values for neurophysins and 1 and comparison with values derived from titration data [5] (see Table 1). n spite of the difficulty in controlling temperature during electrofocusing, the values of p obtained by both procedures are in good agreement. The intrinsic dissociation constants, Kint, for the free /3 and y carboxyl groups of proteins can be of structural interest since it may signal the presence of groups with anomalous dissociation behaviour due to, for example, specific molecular environments. We have used the p values to calculate PKint for neurophysins and 1 using the relation where OL stands for the fraction of dissociated b, ycarboxyl groups, i.e. OL = T/(rzar), where n is the total number of /, ycarboxyl groups in the molecule, r is the number of &amino and guanidinogroups, and a is the analytically determined number

6 Vo1.28, No.1,1972 V. PL~KA, J. F. MCELVY, and H. SACHS 115 of amide residues per molecule of the protein. The values of n and r for neurophysin 1 were taken from a recently published full sequence 1121, for neurophysin from the amino acid composition data [2,5]. n the latter case the uncertainty of the calculated values includes possible errors of amino acid and ammonia analysis as well as of p estimation. n the most unfavorable case all of these errors participate in the result of the calculation (no compensation of errors is to be expected). Using the appropriate formula [13] the maximal error of pkint may be calculated as and, after calculating partial differential quotients, (3) where f stands for the righthand side of Eq. (l), d (x) is the absolute error of variable 2. The standard deviation of p is apparent from Table 1. The errors in n, r, and a are generally accepted to be about 5 Ole. For neurophysin (n = 17, r = 7, a = 6; see [2]) the value of PKint will lie within the broad interval 4.19 f For calculations based on a known sequence the errors in n and r are zero ; for neurophysin 1 accordingly the maximal error is only that of p and pkint = 4.32 f An earlier value [5] of PKint = 4.44 for this protein was based on the data from amino acid analysis, which indicated a slightly different number of arginine residues. The differences in the values of PKint for neurophysin and 1 are of the same magnitude as the lower of both errors and cannot therefore be considered significant. Both values lie within the range for normal sidechain carboxyl groups. We are most appreciative of our helpful discussions with Professor J. Rudinger and Dr Ester Breslow. The generous help of Mr. A. V. Schwally, LKB engineer, is gratefully acknowledged. REFERENCES 1. Hollenberg, M. D. & Hope, D. B. (1967) Biochem. J. 104, Rauch, R., Hollenberg, M. D. & Hope, D. B. (1969) Biochem. J. 115, Hollenberg, M. D. & Hope, D. B. (1968) Biochim. J. 106, Abrash, L. S. (1966) Ph. D. Thesis, Cornell University, New York. 5. Breslow, E., Aanning, H., Abrash, L. & Schmir, M. (1971) J. Biol. Chem. 246, Folin, 0. & Ciocalteau, V. (1927) J. Biol. Chem. Y3, 627. Catsimpoolas, N. (1968) And. Biochem. 26, 480. Wrigley, C. W. (1968) J. Chromatogr. 36, 362. Ornstein, L. & Davis, B. J. (1962) Disc Electrophoresis, Distillation Products ndustries, Rochester, New York. Brown, W. D. & Green, S. (1970) Anal. Biochem. 34,593. Nilsson, P., Wadstrom, T. & Vesterberg, 0. (1970) Biochim. Biophys. Acta, 221, 146. Walter, R., Schlesinger, D. H., Schwartz,. L. & Capra, J. D. (1971) Biochem. Biophys. Res. Commun. 44,293. Scarborough, J. B. (1966) Numerical Mathematical Analysis, pp. 810, The John Hopkins Press, Baltimore. PliBka, V., Barth, T. & Thorn, N. A. (1971) Acta Endocrinol. 67, 1. V. Pliika s present address: nstitut fur Molekularbiologie und Biophysik E. T. H. ZiirichHonggerberg, CH8049 Ziirich, Switzerland J. F. McKelvy s present address: Department of Anatomy, School of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06032, U.S.A. H. Sachs Roche nstitute of Molecular Biology Nutley, New Jersey 07110, U.S.A. 8*