Purification of bovine growth hormone and prolactin by preparative electrophoresis

ANALYTICAL

BIOCHEMISTRY

Purification Prolactin U. J. LEWIS, Di&ion

of

24, 162-175 (1968)

of Bovine Growth Hormone and by Preparative Electrophoresis’ E. V. CHEEVER,

AND

B. K. SEAVEY

Scripps Clinic and Research California 9%X?7

Endocrinology,

Foundation,

La Jolla,

Received October 2, 1967

Polyacrylamide gel used either in slab form (1) or as a column (2-4) has proved to be an excellent medium for electrophoresis. The high degree of resolution which can be obtained on an analytical scale has been achieved on a preparative scale with varying degrees of success (4-10). Pituitary growth hormone and prolactin have been shown to contain a number of components (11, 12) when analyzed by gel electrophoresis. During our work with these multiple components, we found that a commercially available preparative electrophoresis instrument2 could be used very effectively for the isolation of these substances. This communication summarizes our experience with electrophoretic fractionation of purified hormone preparations and also the isolation of growth hormone and prolactin directly from a pituitary extract.3 MATERIALS

AND

METHODS

Bovine growth hormone used as starting material for electrophoretic purification was isolated from the pituitary gland by a method previously described (13) and further purified by the procedure of Dellacha and Sonenberg (14) on Sephadex G-150. Bovine prolactin was isolated by acid-acetone extraction of pituitary glands (15) Hormone Preparations.

‘This work wan supported by U. S. Public Health Service Grants K3-AM-3760 and AM-09537 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, and by Grant P-429 from the American Cancer Society. 2Buchler Poly-Prep, Buchler Instruments, 1327 Sixteenth Street, Fort Lee, New Jersey. This instrument is based on the design of Jovin et al. (7). 8We wish to thank the Endocrinology Study Section of the National Institutes of Health for the samples of bovine growth hormone (B-12) and prolactin (B-1). The growth hormone had a unitage of 0.97 USP unit/mg and, prolactin, 13 IU/mg. 162

PREPARATIVE

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163

and then further purified by chromatography on Sephadex G-150 (16) using 0.05 M N&HCO, as a buffer (17). Bovine pituitary extract was prepared by homogenizing anterior lobes with 0.05 M sodium carbonate buffer, pH 10 (5 ml buffer/gm tissue), stirring 2 hr at 5”, and centrifuging at 30,000 g for 1 hr. The supernatant fluid was saved and passed through a column of Sephadex G-150 (17) to remove only the unretarded material of the first peak. All remaining proteinaceous material eluted from the column was combined and concentrated by lyophilization. Preparative and Analytical Electrophoreeis. The Buchler Poly-prep’ fitted with a porous glass membrane was used in all the studies reported. The cross-sectional area of a column of gel prepared in this instrument was 15.8 cm2. The Tris-glycine buffer system of Ornst,ein (2) and Daris (3) as described by Jovin et al. (7) was used. The column was maintained at 5” by means of circulating water. The height of the column of gel and other electrophoretic variables, together with the method of application of sample, are discussed in detail in the section on “Results.” The optical density (280 mp) of the effluent was monitored by means of a flow eel1 (LKB Uvieord II) and recorder. A fraction collector was set to collect 2 ml volumes. Desired fractions were dialyzed against 0.05 M NH,HCO,, for 24 hr and then lyophilized. Analytical electrophoresis at an alkaline pH (9.5) was carried out with the same buffer system employed with the preparative instrument. An 0.5 X 5 cm running gel made with 7.570 monomer was used. Also used for analytical work was the acidic system of Reisfeld et al. (18’1. For analytical purposes 50-100 pg of sample was applied. The gels were stained with amido black 1OB. lodination. Preparations were labeled with In11 according to the method of McConahey and Dixon (19). The uptake of Is11 was approximately 40%. Samples were counted in a well-t.ype scintillation spectrometer as described previously (17). Immunodiflusion Tests. Preparation of antisera and procedure for the double diffusion method has been described (17). The growth hormone and prolactin iised as antigens contained all the multiple components discussed in this communication. The volume of antiserum or sample per well was 10 ~1. NH,-Termin’al Amino Acid Analysis. The fluorodinitrobenzene method as described by Fraenkel-Conrat et al. (20) was used. Bioassays. Growth hormone was assayed by the method of Greenspan et al. (21) using 10 animals per group. Prolactin was determined by the pigeon crop sac method’as described by Nicoll (22) using 12 birds for each assay. Reference material was injerted over one side of the crop

164

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SEAVEY

and test substances over the other side. Mucosa scraped from an area 4 cm in diameter was weighed (dry). RESULTS Fractionation

of Prolac tin

See Table 1 for summary of data. An electrophoretic pattern of the prolactin that was fractionated by preparative electrophoresis is shown in Figure 2. Roman numeral designations will be used in describing the components. For example, the major, slowest migrating band will be referred to as prolactin-I. CQncentra~ti6n

and Type

of Monomer

Used

for

the Running

Gel.

Previously (23) a running gel that was 7.5% in monomer (acrylamide) was’used for the separation of buffer-front material from the multiple components of prolactin. The system was ideal for this type of separation but gave poor resolution of the components of prolactin. Knowing that the multiple components differed essentially in charge only (see “Discussion”), we substituted a lower gel which was 4% in monomer for the usual 7.5% acrylamide gel in order to obtain a faster rate of migration for the prolactin and therefore have less band spreading due to diffusion. A gel made of 4% acrylamide and 0.2% N,N’-methylenebisacrylamide did not collapse and block the elution chamber even for runs which lasted 10 hr if the temperature was maintained near 5°C. For fractionations at room temperature this type of gel was unsatisfactory because it tended either to fall or to swell and obstruct the elution chamber. This fault, could be overcome if the gel was formed on top of a small-‘base of 7.5% acrylamide. The base was usually 10-15s of the entire column. No loss of resolution occurred; in fact, when comparable columns were examined on analytical sized columns, band sharpening took place as the protein passed from the 4% gel into the 7.5% gel. This type of band sharpening has been reported (24). We also tried using ethylene diacrylate (25) at a final concentration of 0.5% in place of the N,N’-methylenebisacrylamide. Gel made with this reagent seemed to adhere more tenaciously to the glass and had less tendency to slip. However, it was unsatisfactory for long runs because of swelling of the gel exposed to the buffer passing through the elution chamber. Column Length and Sample Load. For separation of the multiple components of prolactin a column 6.8 cm in height (108 ml of gel) was used. A sample load of 50 mg could be resolved on such a column as shown in elution pattern B of Figure 1. When the column length was decreased bv one half, the resolution was a great deal poorer as shown in Figure 1A.

IB 4A 4c 4D

50 50 15oa 1000

Amount, mg

+ + +

use of spacer

6.5 2.9 2.9 6.8

Column height, cm

TABLE 1 for Fractionation by Preparative

108 54 54 108

Gel VOlUIJX, IlIP %F

50 125 125 50

Electrophoresis

6.5 1.0 1.0 6.5

Time for AF to be &ted, hr

4 4 4 4

Time to f&is~lat~ef

Abbreuiutions: GH, growth hormone; BF, buffer front; +, spacer used; - , no spacer; +, no spacer needed if sample was completely dissolved. Q Protein concentration by method of Lowry et al. (29). b Pituitary extract from which growth hormone was isolated by electrophoresis. c Calculated from 15.8 cm2 cross-sectional area of gel. Running gel was 4% in acrylamide and 0.2y0 in N, N’-methylenebisacrylamide. Flow rate was 1 ml/min. Sample volume was 10 ml in all cases.

Prolactin Growth hormone Pituitary extract (for GH) Extract (for prolactin)b

Sample

Elution pattern in Fig.

Summary of Conditions

B 2

R E 2 % 8

il

if

rf

;%

166

‘LEXhS

j CHEEVER,

AtiD

‘SEA-Y-

To isolate larger quantities of the faster migrating components, the starting material was incubated first at pH 16 to increase the amount af the more acidic bands (Fig. 2) and then fractionated on a column 6.8 cm in length, as shown in the elution pattern D of Figure 1. The nature of the shoulder that appears in the elution patterns just before the buffer front is eluted is unknown. The material in the peak showed no stainable bands when concentrated and analyzed on small columns. Applicatbn of Sample and Use of Large-Pore Gel. A 50 mg sample of prolactin was dissolved in 1 ml of 0.05 M carbonate buffer, pH 10, and 1

A

1

O.S-

a

-

D

-

4

0.4 -

,i

t a 3 E

0.8 -

4

0

0.4.

0

0

1

2

3

4

5 0 TIME (HOURS)

1

2

3

4

FIQ. 1. Elution diagrams for electrophoretic purification of prolactin: (A) 50 mg of multiple component prolactin used, no spacer, 3.4 cm running gel of 4% acrylamide, 50 mA, 1 ml/min buffer flow rate. (B) Same as A except that a 6.8 cm running gel was used. (C) Same column conditions as A; sample was material of peak 4 of pattern B which had been kept in solution for 4 days before rerunning (see text for details). (D) Same column conditions as B; sample was 50 mg of incubated protein. In all samples, zero time was taken as the point when buffer front peak (peak 1) was maximum. Shaded areas represent the portions that were combined for tests.

diluted to 10 ml with the same Tris-H,PO,, buffer used in the large-port gel (spacer and sample areas) of the disc electrophoresis method (2, 3’. The solution was made 5% in sucrose and layered on the running \rc:l. Current and Flow Rub. Using a column 6.8 cm in height, a current of 50 mA, and a flow rate through the elution channel of 1 ml/min gave the results shown in Figure lB, C, and D. No temperature rise in the upper buffer compartment was detected when the circulating cooling water (4 liters/min) was maintained at 5”. Increasing the current to 75 mA caused approximately a lo rise in temperature.

PREPARATIVE

167

ELECTROPHORESIS

Recovery of Protein. The most satisfactory way we found of measuring recovery of protein was by adding trace quantities of l”lI-labeled protein to an unlabeled sample before fractionation by electrophoresis. For this type of experiment 49 mg of unlabeled prolactin and 1 mg of labeled hormone were mixed and subjected to electrophoresis. Radioactivity in the elutcd material and in the gel after electrophoresis was measured. A 77% recovery of prolactin was achieved. The unrecoverable radioactivity was distributed between the polyacrylamide gel and glassware. Electrophoretic Homogeneity of Isolated Components. Figure 2 illustrates t,he electrophoretic homogeneity of the prolactin componenLs obtained by preparative electrophoresis. A small amount of (,ht next BOVINE

STARTING MATERIAL

El

PROLACTIN

61’

82

83

EJ4

D”2

0%

D”,

cfs

INC

FIQ. 2. Analytical disc electrophoresis patterns (pH 9.5) of prolactin preparations. The lettering beneath the patterns refers to peaks in the elution diagrams of Figure 1. Patterns were actually obtained with material isolated by conditions described for B of Figure 1. The material of the peaks in D of Figure 1 was indistinguishable from corresponding areas of B except for concentration. The pattern designated IWC was obtained by incubation of the starting material at pH 10 for 16 hr at 37”.

more acidic component was seen in each of the fractions. This was probably the result of deamidation (see ‘LDiscussion”) during dialysis and lyophilization of the sample after elution from the column. Production of faster migrating material during postisolation manipulations is also illustrated in Figure 1C. The starting sample for this elution pattern was a combination of the pooled middle sections of peak 4, Figure lB, of four separate runs. Usually a lyophilized sample of prolactin waa dissolved just prior to electrophoresis, but in this experiment (Fig. 1C) the material was in solution for 4 days (5”) at pH 8.3 before electrophoretic fractionation. It can be sclen t#h:lt, there was a great deal more of the second component (peak 3, Fig. 1C) than would be expected from the analytical pattern (Fig. 2, B4) of similar starting material.

168

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Analytical electrophoresis at pH 4 showed only one band for the isolated components. This was not too meaningful, however, since the components are not well resolved in this electrophoretic system (27). The material of peak 1 of Figure lA, B, and D was brown in color. A reduced pyridine hemochromogen test (26) gave evidence for presence of hematin. Peak 1 material of Figure 1C did not contain this colored substance. Homogeneity of Components by Irnmunodiffusion. The prolactin used as starting material contained neither growth hormone nor serum proteins when tested by immunodiffusion. For these tests 50 pg of hormone was used. All three components of prolactin (peaks 2, 3, and 4, Fig. 1B) gave precipitin lines when tested at 0.25 p,g/well. Bioassay. The material of peak 4, Figure lC, was compared with a reference sample of bovine prolactin (P-B1)3 by means of the pigeon crop sac assay. Using the mucosal weight assay of Nicoll (22), the reference material gave a weight of 8.1 -+ 0.7 mg whereas the prolactin-I produced a mucosal weight of 12.2 + 1.4 mg. NH,-Terminal Homogeneity. The principal NH,-terminal amino acid of the starting material was threonine but there was also about 3.5% contaminating NHz-terminal amino acids, as judged by optical density values of the DNP-amino acids4 eluted from the chromatogram. Glutamic acid, aspartic acid, serine, alanine, and phenylalanine were detected. After preparative electrophoresis these NH,-terminal amino acids were no longer seen. Fractionation of Growth Hormone See Table 1 for summary of data. An electrophoretic pattern of the preparation of growth hormone that was fractionated by electrophoresis is shown in Figure 4. The Roman numeral designation will be used to identify the various components. Concentration and Type of Monomer for the Running Gel. As with prolactin, we had evidence (see ‘LDiscussion”) that the multiple components of growth hormone differed principally in charge. Excellent resolution was obtained, therefore, with a running gel made with 4% acrylamide. This concentration of monomer together with 0.2% N,N’methylenebisacrylamide was used in all studies reported. The observations made concerning the stability of this type of gel when used for purification of prolactin also apply to the fractionation of growth hormone. Column Length, Current, anad Flow Rate. Bovine growth hormone has a lower electrophoretic mobility than prolactin (compare patterns of the 4 DNP

= dinitrophenyl.

PREPARATIVE

ELECTROPHORESIS

I69

two proteins in Figures 2 and 4). When an attempt was made to isolate growth hormone using the same column conditions as for prolactin, the cluted components were too diffuse to be of value. Approximately 14 hr was required before the slowest migrating band appeared. Even decreasing the column length to 2.9 cm was of little value when the current was kept at 50 mA, as is shown in Figure 3B. When the current was increased to 125 mA with the 2.9 cm column, a satisfactory elution pattern was obtained (Fig. 3A). The temperature in the upper buffer compartment

TIME (HOURS1

Fm. 3. Elution diagrams for electrophoretic llrolactin: (A) 50 mg of multiple-component gel of 4’,; acrylamide, 125 mA, 1 ml/min except 50 mA used. (C) 150 mg pituitary 100 mg of material of peaks l-5 shown in 1 ml/min flow rate. In all rases, zero time front elution peak (peak 1) was maximum. caomhinrd for tcsta.

purification of growth hormone ant1 growt,h hormone, spacer, 2.9 cm running flow rate. (R) Samr condit,ions as in .I extract, column conditions as in A. (D) C, no sparer, 6.8 cm running gel, 50 mA, was taken as the point when the buffer Shndrd arras represent thr port,ion

increased 3” but this did not appear to distort the bands during clcctrophoresis. The flow rate through the elution channel was 1 ml/min. Application of Sample and Use of Large-Pore Gel. Growth hormone was not completely soluble at pH 6.7, the pH of the buffer of t’he largepore gel. The sample could be used with this amount of t#urhidit.y if :I spacer was employed. Alternately, the insoluble portion could 1,~ removed by centrifugation and the clear solution mixed with 5% sucrose and layered on the running gel. Tn the dat,a presented, 50 mg of growth hormone was dissolved in 1 ml of 0.05 M carbonate buffer, pH 10, dilutclcl

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SEAVEY

to 10 ml with the Tris-H,PO, buffer used in the large-pore gel, and made 5% in sucrose. The turbid solution was then layered upon a 10 ml spacer. Recovery. Using trace quantities of 1311-labeled hormone mixed with unlabeled carrier, we calculated the recovery to be about 69%. The value was lower than that obtained with prolactin because losses took place by entrainment in the spacer or during removal of insoluble material when centrifugation was used to clarify the starting sample. Electrophoretic Homogen,eity. Figure 4 shows that the two slowest migrating components (growth hormones-I and -II) could be obtained essentially free of faster migrating material, whereas the more acidic BOVINE

STARTING MATERIAL

Al

Al’

GROWTH

A2

HORMONE

A3

FIG. 4. Analytical disc electrophoresis patterns preparations. The lettering beneath the patterns diagrams of Figure 3. The pattern designated INC the starting material at pH 10 for 16 hr at 37”.

A4

A5

INC

(pH 9.5) of growth hormone refers to peaks in the elution was obtained by incubation of

components were contaminated with the next fastest moving band. These more acidic components could be obtained in the same degree of purity as the first two components if the growth hormone was first incubated at pH 10, 16 hr, in order to increase the concentration of the more acidic bands (pattern in Figure 4). A column 4.4 cm in height was then used to separate the components. The starting material contained two weakly staining components (marked with double arrow in Figure 4). These contaminants appeared in the buffer front peak (peak 1, Fig. 3A) and could be detected by analytical electrophoresis (Fig. 4, Al). Analytical electrophoresis at pH 4 to test for homogeneity was unsatisfactory. Not only were the components poorly resolved in this system, but the hormone did not migrate well at this pH (28). Homogeneity by Immunodiffusion. Contamination by prolactin of the

PREPARATIVE

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ELECTROPHORESIS

growth hormone used as starting material could bc detected when 15 pg was tested by immunodiffusion. Serum protein also could be shown to be present if 50 pg of the preparation was used. After purification by clectrophoresis, these contaminants were absent from growt,h hormone-I when 50 pg was tested. The contaminating prolactin and serum proteins were found in the buffer front peak (Fig. 3A). Thr three principal components of the growth hormone produced l)rc~c+pit.in lines and all were equally cffe&ive. Kiotrssntj. The material of peak 5, Figure 3A, wab coniparrd w&h :1 reference sample of bovine growth hormone (B-12) .3 The purified material was rss:rJntiaIly of the same potency as the reference material. The :inimals that received saline had tibia1 lint: width of 98 t 5 p; the stanclarrl (25 ,.~g) increased this to 122 k 7 ,J whereas the growt,h hernlone-T (2.5 ELM) produced a tibia1 line width of 132 f 7 ;t.. BOVINE

PITUITARY

EXTRACT

OH HB PROL ALB

STARTING MATERIAL

C1

c2

CS

c4

c5

C6

FIG. 5. Analytical disc clectrophoresis pattern,< (pH 0.5) of fraciiona isolated from :I piktary extract. The lettering beneath the pnt,tvrna rc~fvrs to ~w:~-cs in the elution diagram of Figure 3. GH, qowth hormone ; HI), hrmonlobin; l’rol, lnvl:u*tin: .IIb, albumin; BF, buffer front.

lVH,-Ternkal Homogeneity. The growth hormone usccl as starting material contained mainly alanine -and phcnylalanine RS NH,:terminal amino acids. They were present in approximately equal molar amounts. Small amounts (about 5%) of other NH,-terminal amino acids WCI’C found. These were not detected in thr clectrol~horc~ticnll~ purified material (growth hormone-I). Fractionation

of Pituitary

Extract

See Table 1 for summary of data. An electrophoretic pattern of the pituit:Lry cstr:\vt that, was fract.ionnted by electrophoresis is shown in Figure 5. Idclltifiration of the variolls romponenk of the extract has been described (17).

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C01wwn Conditions. The same sized column, concentration of monomer, current, and flow rate used for the purification of growth hormone were satisfactory for isolation of growth hormone from the extract. A sample of 150 mg of extract protein (29) was dissolved in 1 ml of 0.05 M sodium carbonate buffer, pH 10, diluted to 10 ml with the Tris-H,PCa buffer used in the large-pore gel and made 5% in sucrose. The sample was layered on a 10 ml spacer. As seen in Figures 3C and 5, albumin, prolactin, and hemoglobin were eluted immediately after the buffer front. The growth hormone appeared as a quite homogeneous band after 4 hr (Fig. 3C, peak 6). The prolactin in the pituitary extract could be isolated by subjecting the material of peaks 1-5, Figure 3C, to a second electrophoresis on a column of the type used to fractionate purified prolactin (Fig. 1B). The material first was dialyzed against 0.05 M NH,HCO,, lyophilized, and reconstituted in Tris-H,PO1 buffer before a preparative run. The elution pattern is shown in Figure 3D and analytica eIectrophoretic (pH 9.5) patterns are pictured in Figure 5. The peak indicated by 1 in Figure 3D was composed mainly of buffer-front components, 2 was albumin, 3 was prolactin-II, 4 was prolactin-I, and 5 was hemoglobin. Howwgeneity of Compon,ents. Figure 5 (pattern C6) shows that the growth hormone isolated from the extract (Fig. 3C, peak 6) showed no other component when analyzed by electrophoresis (pH 9.5). The material was free of serum proteins when tested at 2.5 pg by immunodiffusion. Prolactin was also absent when 50 pg of this preparation of growth hormone was tested by immunodiffusion. The prolactin isolated from the extract (Fig. 3D, peak 4) was free of growth hormone and serum proteins as judged by electrophoresis (pH 9.5) and immunodiffusion. However, serum protein was detected in peak 3 of Figure 3D when tested by immunodiffusion at 5 mg/ml. Bioassay. The growth hormone and prolactin isolated from the extract were comparable in biological activity to the reference material and to the growth hormone-I and prolactip-I isolated from partially purified starting material. Alteration

of Elactrophoretic

Mobility

by Iodination

In an earlier paper (23) we reported that the amount of radioactivity in the various bands of a stained disc electrophoresis pattern of lslIlabeled prolactin corresponded to the intensity of the staining. With growth hormone, however, there was no such correlation. The second band of growth hormone (growth hormone-II) had an unusually large amount of radioactivity in relation to its staining properties. Using the

PREPARATIVE

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ELECTROPHORESIS

preparative electrophoresis apparatus, we have been able to obtain additional data that help explain these variations in distribution of radioact.ivity. A mixture of 1 mg of labeled and 49 mg of unlabeled hormone was fractionated by preparative electrophoresis and both optical density (280 rnpl and radioactivity of the eluted material were measured, as shown in Figure 6. Both growth hormone and prolactin wcw 1

GROWTH

I

I

I

HORMONE

PROLACTIN 13-

,2

CPM 00 --_-

12-

-1.3 ---CPM OD -

- 1.2

ll-

-1.1

lo-

- 1.0

9-

-0.9

8..

-0.8

7-

-0.7

0 : il Fr

-0.6

;

-0.5

5

-0.4 -0.3 -0.2 -0.1 .o TUBE NUMBER

TUBE NUMBER

FIG. 6. Distribution of radioactivity in elution diagrams of prolactin and growth hormone. For each protein, 1 mg of “‘I-labeled hormone was mixed with 49 mg of unlabeled hormone. Prolactin w&s fractioned on a column aa desrrih~~d in Figure lH, growth hormone as described in Figure 3A.

examined in this manner. Since only a small amount of labeled hormone: was used in comparison to the amount of unlabeled protein, the optical density curve is representative of unlabeled hormone. For prolactin the optical density and radioactivity curves were close but not exactly superimposable. A much greater discrepancy was found in the case of growth hormone, where the major peak of radioactivity was almost in the same position as component II of growth hormone. These results indicate that t,he hormones had become more acidic upon iodination. Although all components had assumed a more negative charge, the relative amount. of radioactivity in the various peaks still corresponded quite closely to optical density values of the unlabeled hormone. The small variations along the radioactive curve of growth hormone were reproducible but, Ive have no explanation for them.

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DISCUSSION

The disc electrophoresis patterns of bovine prolactin and growth hormone show one principal band and one or more minor bands which migrate ahead of the major component. This is illustrated in Figures 2 and 4 where the components are designated by Roman numerals. The components have been the subject of investigation by a number of workers (28, 30-32). Upon incubating the hormones at pH 10 for 16 hr at 37” the number and intensity of the faster migrating components increased, as illustrated also in Figures 2 and 4. During this alteration (27,33) ammonia is liberated, no change in NH,-terminal groups occurs, and no inhibitor for the reaction was found including dry heat (105”) for 16 hr. After oxidation of prolactin by performic acid (27), liberation of ammonia still occurred upon incubation at pH 10, which would strongly negate an enzymically catalyzed reaction. We feel, therefore, that the components in the preparations that were to be separated by preparative electrophoresis differed mainly in charge. For this reason the gel made with 4% acrylamide was completely satisfactory for electrophoretic separations, serving principally as an anticonvection medium. The high molecular weight particulate material had to be removed from the pituitary extract prior to electrophoretic fractionation or the sample (in sucrose) would not “stack” properly. Instead, the material underwent a mixing soon after the current was applied. The particulate matter did not have to be removed if the sample was photopolymerized in place. Measurement of the distribution of radioactivity in the elution pattern of growth hormone indicated that all the components become more acidic after iodination and not that growth hormone-II is more heavily labeled than the other components, as previously suggested (23). An increased ionization of the hydroxyl group in the tyrosine residues would readily explain this phenomenon. SUMMARY

Experimental conditions are described whereby the multiple components of bovine prolactin and growth hormone can be isolated by preparative electrophoresis on polyacrylamide using a commercially available instrument. A method is also described for isolation of prolactin and growth hormone directly from a crude pituitary extract. Immunodiffusion, bioassay, and NH,-terminal data are presented for the purified prolactin and growth hormone. Data are presented which indicate that the electrophoretic mobility of bovine growth hormone is altered markedly by iodination. Prolactin is also affected but to a lesser degree.

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REFERENCES

10. 11.

S.. Ann. N. Y. Acad. Sci. 121, 350 (1964). ORNSTEIN, L., Ann. AT. Y. Acad. Sci. 121, 321 (1964). D.WIS, B. J., A~I&. N. I’. Acad. Sci. 121, 404 (1964). . HJERTBN, S., JERSTEDT, S., AND TISELIUS, A., Anal. Biochem. 11, 211 (1965). LEWIS, U. J.. AND CLARK, M. 0.. Anal. Biochem. 6, 303 (1963). .~LTSCHUL, A. M., EVANS, W. J.. CIRNEY, W. B., AND MCCOURTNEY, E. J., Life Sci. 3, 611 (1964). .JOVIN, T., CHRAMBACH, A., AND NAUGHTON. M., Anrtl. Biochem. 9, 351 (1964). M.~IZEL, J., Ans. X. I-. Acad. Sci. 121, 382 (1964). RACXISEX. D.. AND CALVANICO, N., Anal. Biochem. 7, 62 (1964). CATT, Ii., AND MOFFAT, B., Endocrinology 80, 2 (1967). FERGUSDN. K. A., AND WALLACE, A. L. C., Hecerd Pmyr. Hormone Kes. 19, 1

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BBRRETP,

13.

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1. RAYMOND,

2.

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