PURIFICATION OF CANINE PROLACTIN BY PREPARATIVE ISOTACHOPHORESIS

KNIGHT, M. GRONOW AND J. M. HAMILTON Department of Experimental Pathology and Cancer Research, School of Medicine, Leeds, LS2 9NL

P. J.

(Received! July 1975) SUMMARY

Preparative isotachophoresis in polyacrylamide gel using carrier ampholytes as 'spacers' has been used to purify prolactin from canine pituitary extracts. Using Ampholine pH 5\p=n-\8as spacers, a prolactin fraction was obtained which was essentially homogeneous as judged by the criteria of disc electrophoresis and isoelectric focusing. Amino acid analysis indicated a close similarity between canine and ovine prolactin. A growth hormone fraction, identified by its electrophoretic mobility, was also obtained although this was heterogeneous in disc electrophoresis at alkaline pH. INTRODUCTION

The role of prolactin in mammalian physiology and pathology is receiving increasing atten¬ tion. Since the dog is used extensively in medical research, it would be appropriate to investigate the role of prolactin in this animal. Consequently it is important to obtain purified samples of prolactin to facilitate such studies. Conventional techniques for the purification of polypeptide hormones of the anterior pituitary usually involve gel filtration followed by ion-exchange chromatography. These methods are somewhat time-consuming and each Chromatographie step involves loss of material. Preparative disc electrophoresis has been used for the isolation of prolactin and growth hormone (Groves & Sells, 1968; Lewis, Cheever & Seavey, 1968; Gala, 1972) but has the disadvantage that in the final stage, where zone electrophoresis takes place, there can be considerable spreading of the protein bands by diffusion. Isotachophoresis (Haglund, 1970), which is here defined as steady-state stacking (Ornstein, 1964) in the presence of spacer ions, has the theoretical advantage that diffusion is inhibited throughout the whole of the

separation. Adjacent protein zones are kept apart by non-u.v.. absorbing ampholytes (Ampholine, LKB) (Vesterberg & Svensson, 1966), hence detection of protein zones in the final elution pattern is facilitated. On this basis preparative isotachophoresis (ITP) was applied to the purification of canine pituitary prolactin. METHODS

obtained frozen on solid C02 from Pel-Freez Biologicals Inc., pituitaries Arkansas 72756, U.S.A. To remove a major part of the blood proteins and growth Rogers, hormone, the pituitaries were homogenized in a loose fitting all-glass homogenizer in cold 0-15 M-saline, pH 5-5, at 10 ml/g tissue and immediately centrifuged at 50000 g for 1 h. The supernatant was dialysed against distilled de-ionized water and lyophilized. The residue was extracted in an identical manner with fresh saline, centrifuged and the supernatant dialysed

Canine

were

lyophilized. To extract prolactin, the residue was homogenized in a tight-fitting Teflonglass homogenizer in 005 M-carbonate buffer (Lewis, Singh & Seavey, 1972), pH 9-5, at 10 ml/original g tissue and stirred for 3 h at 4 °C before centrifugation at 50000 g for 1 h. The supernatant was dialysed and lyophilized and the residue discarded. To minimize enzymic breakdown of the prolactin, the saline and carbonate solutions contained 10 mM-pand

aminobenzamidine (Ellis, Nuenke & Grindeland, 1968). Analytical disc electrophoresis was performed using a Shandon polyacrylamide gel electrophoresis apparatus. The buffer system was that described by Davis (1964) using 7-5 % acrylamide gel rods. Preparative ITP was performed in the LKB 7900 Uniphor (LKB Produkter, Sweden). The apparatus was assembled and buffers and acrylamide solution prepared according to the instruction manual supplied by LKB. Fifty millilitres of 7-5 % acrylamide solution was photopolymerized in the Uniphor column and the remainder of the column was filled with dilute terminating buffer. The sample solution consisted of 90 mg of the dialysed lyophilized carbonate extract of canine pituitaries dissolved in 15 ml terminating buffer plus 1-5 ml Ampholine (40 %, w/v), pH 5-8. This solution was layered onto the gel column with the aid of a syringe and a length of Polythene tubing. With the lower electrode as the anode, a current of 12 mA, 1000 V, was passed for 30 h. The elution buffer (dilute leading buffer) was monitored by % transmittance at 280 nm in an LKB Uvicord II. Fractions were collected at 3 ml/tube, 18 ml/h. Pooled fractions were dialysed against 0-2 M-NaCl for 36 h, followed by distilled deionized water for 48 h, and lyophilized. The biological activity of the fraction containing prolactin was determined by the method of Nicoli (1967). For amino acid analyses, hormone was dissolved in 1-0 ml 6 M-HC1 containing 0-15 % phenol, deoxygenated in vacuo and flushed with nitrogen. The sample vials were sealed and hydrolysis carried out at 110 CC for 24 h. The HC1 was removed in vacuo over NaOH pellets before analysis on a Bio-Cal BC-100 Analyser using a single column system. RESULTS

Figure 1 shows a comparison of the proteins contained in the initial saline extract with those removed by the subsequent extraction with carbonate buffer. The identity of albumin, growth hormone by electrophoretic mobility and prolactin by bioassay has been established previously (Saluja, Gronow & Hamilton, 1973). The first saline extract contained the majority of the blood proteins together with a considerable amount of growth hormone. Little prolactin was extracted by the saline as can be seen from the absence of a marked prolactin band in the electrophoretogram. The second saline extract removed qualitatively the same material though in reduced amounts, while the carbonate buffer at pH 9-5 removed residual growth hormone and the prolactin. Re-extraction of the residue after this step did not remove any additional prolactin. The band labelled D-Pr in Fig. 1 was believed to con¬ sist of deamidated prolactin. Prolactin molecules which have lost an amide group show increased mobility towards the anode in disc electrophoresis at alkaline pH (Lewis, Cheever & Hopkins, 1970; Gráf, Cseh, Nagy & Kurcz, 1970). However, the possibility that this band may be the result of proteolysis is not ruled out. Carbonate extract (90 mg), the residue from 2 g of pituitaries, was subjected to prepara¬ tive ITP. The resulting elution profile is shown in Fig. 2. The first peak, tubes 72-80, con¬ tained u.v.-absorbing material which was non-proteinaceous in nature. The remaining tubes were divided into seven fractions on the basis of the elution profile and were dialysed and

lyophilized.

Fig. 3 shows that the proteins contained

in fractions

3, 4 and 7

were

identical electro-

phoretically

with deamidated prolactin, prolactin and growth hormone, respectively. It that fraction 4, which contains the canine prolactin, is homogeneous in disc electrophoresis at this pH. Analysis of the material by isoelectric focusing showed it to be more homogeneous than a 30 i.u./mg commercially available preparation of ovine prolactin

can

be

seen

(not shown). Pigeon crop-sac bioassay results showed that the ITP fraction 4 (canine prolactin) had a potency of 15-16 i.u./mg, while ITP fraction 3 (deamidated prolactin) had a potency of 10

i.u./mg.

Amino acid analysis showed that the total weight of amino acid residues accounted for only 50 % of the dry weight of ITP fraction 4 applied. The constitution of the remaining 50 % of fraction 4 is unknown, although it is possible that it represents residual Ampholine. Table 1 gives the amino acid analysis of canine prolactin compared with the published data for ovine prolactin (Li, Dixon, Lo, Schimdt & Pankov, 1970).

1. (A) 200 /tg lyophilized saline extract of canine pituitary glands compared with (B) 200 /tg of the subsequent carbonate extract. Disc electrophoresis in 7-5 % acrylamide gel rods, pH 9-3. Abbreviations: ALB, albumin; BF, buffer front; D-Pr, deamidated prolactin; GH, growth

Fig.

hormone; Pr, prolactin.

DISCUSSION

Polyacrylamide gel ITP has been examined on both preparative (Clemmensen & Svendsen, 1973; Svendsen, 1973) and analytical (Chrambach, Kapadia & Cantz, 1972; Griffith & Catsimpoolas, 1972) scales and the theoretical considerations discussed. On a preparative scale, successful separations of multicomponent protein systems have been achieved

20 30

40

I

~l· 6 i-

f-

50 60

70 SO

90

72

80

90

100

110 120 Tube number

130

140

150

160

Fig. 2. Elution profile from preparative isotachophoresis of 90 mg carbonate extract of canine pituitary glands. Elution rate was 18ml/h; 3 ml/tube. Yields of hormone-containing fractions: fraction 3, 50 mg; fraction 4, 60 mg; fraction 7, 6-7 mg.

Table 1. Amino acid composition

of ovine and canine prolactins Molar ratio

Amino acid Ala

Arg

Asx

Cys (i) Glx

Gly

His He Leu

Lys Met Phe Pro

Ser Thr

Trp Tyr Val

Ovine* 9 11 22 6 22 11 8 11 22 9 7 6 11 15 9 2 7

10

Caninef 13 13 22 24 11 7 11 22 9 1 6 8 15 6

6 11

* Data from Li et al. (1970). t Mean experimental results from duplicate amino acid analyses given Cysteine and tryptophan were not measured.

to the nearest whole

number.

(Clemmensen & Svendsen, 1973). The present work confirmed the usefulness of preparative ITP (Svendsen, 1973) in isolating single components from protein mixtures. The separation of prolactin from a carbonate extract of canine pituitaries was successfully achieved and contamination by deamidated prolactin was minimal.

Fig. 3. Disc electrophoretic analysis of fractions 3,4 and 7 from isotachophoresis of 90 mg carbonate canine pituitaries. Fractions 3 and 4 contained deamidated prolactin and prolactin, respectively, while fraction 7 contained growth hormone plus several faster-moving bands. Condi¬ tions and key to abbreviations as in Fig. 1. extract of

prolactin isolated by ITP was contaminated to the extent of 50 % of the dry weight by a non-proteinaceous material, possibly Ampholine. However, that did not appear to have altered the properties of prolactin since the electrophoretic mobility of the discreet prolactin band was the same as that of prolactin before ITP. Similarly, in the pigeon cropsac assay, ITP fraction 4 gave a parallel dose-response curve to the standard ovine prolactin. However, as only 6 mg of this material was obtained, removal of the non-proteinaceous contaminant was not attempted. The amino acid analysis shown in Table 1 was based on the assumption that ovine and canine prolactins have similar molecular weights. Bearing this in mind, the analysis showed an interesting conformity between the two hormones. The most striking difference was in the methionine residues: only one residue per molecule in the canine hormone compared with seven in the ovine hormone. Care was taken to eliminate oxygen from the hydrolysis mixtures though it is possible that slight oxidation losses were incurred. Low levels of methionine (2 residues/molecule) have been reported for rat prolactin (Ellis, Grindeland, Nuenke & Callahan, 1969). Under the conditions used in this experiment, prolactin was separated from its more anodal form whereas the growth hormone, identified by its electrophoretic mobility, was heterogeneous in this respect. This may be due to the appreciable difference in pi between the two hormones which has been measured at 6-6 for canine prolactin and 7-7 for canine affects the ability growth hormone (Saluja et al. 1973). The choice of Ampholine pH range & Catsimpoolas, to separate protein mixtures by ITP (Chrambach et al. 1972; Griffith middle of the to close the range lies of the Ampholine this case in and prolactin pi 1972) where one would expect the greatest concentration of ampholytes. The pi of growth hormone, on the other hand, lies near the upper limit of the Ampholine range where the concentration of ampholytes would be lower. This may account for the greater heterogeneity The canine

of the

growth hormone since there may not have been enough ampholytes to keep adjacent protein zones apart. However, when designing ITP separations it should be remembered that pi is not the only factor governing mobility in the support medium, gel-pore size and molecular weight of the proteins should also be taken into account. The assistance of LKB Instruments Ltd. is gratefully acknowledged, in particular that of Mr D. Fairley. The bioassay was performed by Dr I. A. Forsyth, NIRD, Shinfield, Reading, and the amino acid analysis was performed by the Joint Sequencing Unit of Leeds Univer¬ sity. This work was supported by a grant from the Yorkshire Council of the Cancer Research

Campaign.

REFERENCES

Chrambach, ., Kapadia, G. & Cantz, M. (1972). Isotachophoresis

on polyacrylamide gel. Separation Science!, 785-816. Clemmensen, I. & Svendsen, P. J. (1973). Isolation of the plasmin resistant E-antigenic fibrinogen breakdown product by isotachophoresis. Science Tools 20, 5-6. Davis, B. J. (1964). Disc electrophoresis. II. Method and application to serum proteins. Annals of the New York Academy of Sciences 121, 404—427. Ellis, S., Grindeland, R. E., Nuenke, J. M. & Callahan, P. X. (1969). Purification and properties of rat prolactin. Endocrinology 85, 886-894. Ellis, S., Nuenke, J. M. & Grindeland, R. E. (1968). Identity between the growth hormone degrading activity of the pituitary gland and plasmin. Endocrinology 83, 1029-1042. Gala, R. R. (1972). Studies on the purification of rat prolactin from anterior pituitary organ culture by preparative disc electrophoresis. Hormones 3, 343-353. Gráf, L., Cseh, G, Nagy, I. & Kurcz, M. (1970). Evidence for deamidation of prolactin monomer. Acta Biochimica et Biophysica Academia Scientiarum Hungaricae 5, 299-303. Griffith, A. & Catsimpoolas, N. (1972). General aspects of analytical isotachophoresis of proteins in poly¬ acrylamide gels. Analytical Biochemistry 45, 192-201. Groves, N. E. & Sells, B. H. (1968). Purification of rat prolactin and growth hormone using preparative polyacrylamide gel electrophoresis. Biochimica et Biophysica Acta 168, 113-121. Haglund, . (1970). Isotachophoresis A principle for analytical and preparative separation of substances such as proteins, peptides, nucleotides, weak acids, metals. Science Tools 17, 2-13. Lewis, U. J., Cheever, E. V. & Hopkins, W. C. (1970). Kinetic study of the deamidation of growth hormone and prolactin. Biochimica et Biophysica Acta 214, 498-508. Lewis, U. J., Cheever, E. V. & Seavey, . K. (1968). Purification of bovine growth hormone and prolactin by preparative electrophoresis. Analytical Biochemistry 24, 162-175. Lewis, U. J., Singh, R. N. P. & Seavey, . K. (1972). Problems in the purification of human prolactin. In Prolactin and carcinogenesis, pp. 4-12. Eds A. R. Boyns & K. Griffiths. Cardiff: Alpha Omega Alpha -

Publishing. Li, C. H., Dixon, J. S., Lo, T.-B., Schmidt, K. D. & Pankov, Y. A. (1970). Studies on pituitary lactogenic hormone. XXX. The primary structure of the sheep hormone. Archives ofBiochemistry and Biophysics 141, 705-737.

Nicoli, C. S. (1967). Bio-assay of prolactin. Analysis of the pigeon crop-sac response to local prolactin injection by an objective and quantitative method. Endocrinology 80, 641-655. Ornstein, L. (1964). Disc electrophoresis. I. Background and theory. Annals of the New York Academy of Sciences 121, 321-349. Saluja, P. G., Gronow, M. & Hamilton, J. M. (1973). Measurement of canine pituitary prolactin. Journal of Endocrinology 56, 245-258. Svendsen, P. J. (1973). On the procedure of preparative isotachophoresis. Science Tools 20, 1-4. Vesterberg, O. & Svensson, H. (1966). Isoelectric fractionation, analysis and characterisation of ampholytes in natural pH gradients. Acta Chemica Scandinavica 20, 820-834.