Protein hormones: detection and extraction of functional fomrs from alkaline polyacrylamide gels

Journal of Biochemical and Bioph.vsical Methods, 10 (1984) 173-179 Elsevier

173

BBM 00445

Protein hormones: detection and extraction of functional forms from alkaline polyacrylamide gels Luis S. Haro and F r a n k J. T a l a m a n t e s Biology Board of Studies, Thimann Laboratories, Universi O, of California, Santa Cruz, CA 95064, U.S.A.

(Received 9 July 1984) (Accepted 7 August 1984)

Summary. A method is presented for rapidly staining protein zones (30 min) in alkaline polyacrylamide gels in the absence of fixatives. Native functional proteins are recovered in homogeneous form after excision of the visible zones from the polyacrylamide matrix. Removal of dye from excised zones is facilitated because only the surface of the gel is stained. Biological activity is then recovered from the gel slices by simple diffusion. The technique makes use of the sensitivity of Coomassie Brilliant Blue G-250 for the detection of proteins ( < 1 p~g). Structural variants of prolactin are isolated and recovered by this method. The method is applicable to studies requiring analytical and semi-preparative electrophoresis of proteins, especially pituitary hormones. Key words: gel electrophoresis; prolactin; growth hormone; gel staining: radioreceptor assay: protein purification.

Introduction In order to analyze alkaline polyacrylamide gels after electrophoresis of native proteins, the bands must be visualized. It is normal for the separated proteins to be fixed in the gel to prevent their diffusion and loss during staining and destaining. The common denaturants used are methanol/acetic acid [1,2], isopropanol/acetic acid [3], trichloroacetic acid [4,5] and perchloric acid [6]. Although the staining methods allow the localization of protein bands, the proteins are no longer in their native functional state. If the protein of interest is needed in its native state it is usual to cut the unstained rod or slab gel into segments and then extract and assay the proteins for biological activity [7]. Alternatively a stained gel can be used as a guide [8]. Protein bands of sufficiently high concentration may be localized by direct photometric scanning of unstained gels at 280 and 310 nm [9] or by observing the light and dark 0165-022X/84/$03.00 ~ 1984 Elsevier Science Publishers B.V.

174 bands due to changes in refractive index [10]. Proteins have also been detected in unstained gels by the use of phosphorescence [11,12]. The given methods do not guarantee that the gel will be sliced in the optimum places due to gel expansion, light scattering, poor absorbance of proteins at 280 nm, or because the protein of interest is not a major component. It is preferable to first localize the separated protein bands and then excise the zone of interest. One such method uses tannic acid in staining native proteins to permit protein recovery [13]. The staining of proteins in the absence of chemical fixatives has been described for two-dimensional isoelectric focusing/sodium dodecyl sulfate gels [14]. The use of alkaline polyacrylamide gel electrophoresis to analyze pituitary hormones and their structural variants is extremely widespread. A technique which permits the extraction of functional hormones and their variants from such a matrix is needed, especially since the amount of material available is often minute. In this communication we present a method for the detection and recovery of native proteins from alkaline polyacrylamide gels in the absence of chemical fixatives.

Materials and Methods

Chemicals Both ovine prolactin (oPRL; NIAMDD-oPRL-14) and bovine growth hormone (bGH; NIH-GH-B-18) were gifts of the National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases. Human placental lactogen (hPL; lot 6033) was obtained from ICN Pharmaceuticals. Acrylamide, N, N'-methylenebisacrylamide and Coomassie Brilliant Blue G-250 were from Bio-Rad. All other chemicals were of reagent grade.

Electrophoresis Alkaline polyacrylamide gel electrophoresis (8% total acrylamide concentration with bisacrylamide representing 2.7% of the total monomer) with a separating pH of 8.9 and no stacking gel was carried out as described [15] on a Hoefer DE electrophoresis unit. Gels were cast in cylinders (13 cm x 5 mm i.d.), loaded with protein and electrophoresed at 3 mA per gel.

Protein staining and destaining Gels were stained in a solution of 0.25% ( w / v ) Coomassie Brilliant Blue G-250 in 12% trichloroacetic acid and destained in a solution of 7% acetic acid/10% methanol/83% water. Alternatively, gels were stained in buffers containing 0.3% Coomassie Brilliant Blue G-250 for various times and destained in the same buffer without dye. The following buffers, all at concentrations of 100 mM were used: glycine/HC1, pH 3.0; ammonium acetate/acetic acid, pH 4.0 and pH 5.0; sodium phosphate, pH 6.0 and pH 7.0; Tris-HCl, pH 8.0 and pH 9.0; and glycine/NaOH, pH 10.0. To see the effect of pH on the destaining reaction, gels were stained in pH 4.0 buffer and destained in the above buffers at pH values of 7.0, 9.0 and 10.0.

175

Recovery of oPRL from stained gels Each visible oPRL isoform was excised from the gel with a razor blade. The stained part of each gel slice was cut away from the unstained portion and the proteins subsequently eluted by diffusion into a buffer containing 25 mM TrisHC1/10 mM CaCI2/0.1% bovine serum albumin, pH 7.6, at 5°C for 16 h.

Radioiodination oPRL was iodinated by a lactoperoxidase procedure previously described [16] with some modifications. To 1 mCi carrier-free Na125I (5 ~1) were added 5 /tg prolactin in 10/.tl 0.05 M sodium phosphate (pH 7.5), 1 ~tg lactoperoxidase in 20/~1 0.5 M sodium phosphate (pH 7.5) and 20/~1 of a 1 : 60000 dilution of 30% H202 in distilled deionized H20. After 3 min the reaction mixture was diluted with 300/.tl 0.025 M Tris-HC1, pH 7.5. The diluted tracer was immediately applied to a 1 × 10 cm Sephadex G-50 column, previously equilibrated with 0.025 M Tris-HC1/10 mM CaC12/0.1% bovine serum albumin/0.1% azide, pH 7.6 and 0.4 ml fractions were collected in 12 × 75 mm glass tubes. The radioactivity of the fractions was monitored with a geiger counter and the 2-3 tubes having the greatest radioactivity were pooled and further separated on a 1 × 20 cm Sephadex G-100 column equilibrated with the same buffer used in the previous column. Fractions from the monomer peak were used in the radioreceptor assay.

Radioreceptor assay Lactating rabbit mammary glands were processed as before [16] except that the membranes were not lyophilized but rather were frozen and stored at - 4 0 ° C after resuspending them in 0.025 M Tris-HC1/10 mM CaC12/0.1% bovine serum albumin, pH 7.6 buffer. The assay was performed as before [16] except that the binding reaction was carried out at 25°C for 24 h.

Results and Discussion

The staining of proteins in the absence of fixatives has been described for two-dimensional isoelectric focusing/sodium dodecyl sulfate polyacrylamide gels [14]. This report extends the usefulness of the method to alkaline polyacrylamide matrices. In addition, evidence supporting recovery of biological activity from stained gels is presented. Staining of proteins in the matrix is dependent on pH, with the optimum occurring at pH 4.0 (Fig. 1). Staining is complete after 30 min and its intensity is similar to that of proteins fixed and stained in trichloroacetic acid (Fig. 2). The lower limit of protein detection in a 5 mm diameter column is 1 /~g (Fig. 3). Gels are best destained at alkaline pH (Fig. 4). Prolactin structural variants, presumably des-amido forms [17] exhibit functional activity in a radioreceptor assay after staining and recovery from gel slices (Fig. 5). The purification of protein hormones by the use of PAGE is well documented [18-22]. The present technique improves its usefulness as both a preparative and an analytical tool. It is applicable in the small scale preparative work of structural

176

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Fig. 1. pH dependence of protein staining: The protein hormones bGH, hPL and oPRL were stained with Coomassie Brilliant Blue G-250 dissolved in various pH buffers after being separated on an alkaline polyacrylamide gel. (A) glycine/HCl, pH 3.0; (B) ammonium acetate/acetic acid, pH 4.0; (C) ammonium acetate/acetic acid, pH 5.0; (D) sodium phosphate, pH 6.0; (E) sodium phosphate, pH 7.0; (F) Tris-HCl, pH 8.0; (G) Tris-HCl, pH 9.0; (H) glycine/NaOH, pH 10.0.

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Fig. 2. Time dependence of protein staining, bGH, hPL, and oPRL were stained with Coomassie Brilliant Blue G-250 in trichlororacetic acid or ammonium acetate, pH 4.0 for various times after separation on alkaline polyacrylamide gels. (A) trichloroacetic acid; (B) ammonium acetate, 30 min; (C) ammonium acetate, 1 h; (D) ammonium acetate, 3 h.

177

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~----bGH -3--oPRL hPL i Fig. 3. Sensitivity of stain, bGH, hPL, and oPRL in various amounts were stained 30 min with Coomassie Brilliant Blue G-250 in ammonium acetate, pH 4.0 after separation on alkaline polyacrylamide gels. (A) 0.63 /~g/hormone; (B) 1.25/~g/hormone; (C) 2.5/~g/hormone; (D) 10.0 p,g/hormone.

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Fig. 4. Destaining gels. bGH, hPL, and oPRL were stained with Coomassie Brilliant Blue G-250 in ammonium acetate, pH 4.0 for 5 h after electrophoresis on alkaline polyacrylamide gels and then destained for 5 h in buffers with various values. (A) Gel stained for 5 h; (B) gel destained at pH 7.0; (C) gel destained at pH 9.0; (D) gel destained at pH 10.0.

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dilution factor of oPRLeluate ( o-o, x--~) Fig. 5. oPEL radioreceptor assay. Native oPEL and des-amido oPRL were tested in a radioreceptor assay after staining and elution from polyacrylamide gels; (e) N I A M D D - o P R L - 1 4 standard; ( O ) native oPEL; ( × ) des-amido oPRL.

variants of p i t u i t a r y h o r m o n e s [17,23-26] as well as in studies of n o n - m a m m a l i a n p i t u i t a r y h o r m o n e s that are scarce or u n a v a i l a b l e [27].

Simplified description of the method and its applications Native proteins fractionated in an alkaline polyacrylamide matrix are detected with 0.3% ( w / v ) Coomassie Brilliant Blue G-250 in 100 m M a m m o n i u m acetate/acetic acid pH 4.0 for 30 min and destained in the identical buffer without dye. Visible protein zones are easily excised from the polyacrylamide gel with the use of a razor blade. Stained portions of each protein zone are then trimmed away from the unstained portions. Next, native proteins are eluted from the gel slice by simple diffusion and subsequently tested for functional activity. The present method permits the separation, localization and recovery of proteins in their native state. It is especially useful for recovering the structural variants of pituitary protein hormones that are frequently seen in alkaline polyacrylamide gels. In addition, this technique provides a valuable approach for small-scale preparation of pituitary hormones that are currently scarce, such as non-mammalian growth hormones and prolactins.

Acknowledgements This research was s u p p o r t e d by N I H G r a n t s RR-08132 a n d H D - 1 9 9 6 6 to F.J.T.

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