A three-step method for isolating a few to several hundred milligrams of protein

Journal o f Biochemical and Biophysical Methods, 1 (1979) 171--187 © Elsevier/North-ttolland Biomedical Press

A TtIREE-STEP METHOD HUNDRED MILLIGRAMS

FOR ISOLATING OFPROTEIN

171

A FEW TO SEVERAL

NGA Y. NGUYEN and ANDREAS CHRAMBACH Endocrinology and Reproduction Research Branch, National lnstilule o f Child ttealth and Human Development, National Institutes o f Health, Belhesda, MD 20205, b:S.A.

(Received 3 November 1978; accepted 19 April 1979)

An operationally simple general protein isolation method was devised f:.'om three previously available separation tools, and was tested by application to two demanding fractionation problems and for yield. One test system was the isolation by gel electrofocusing of two model proteins with pl values of 4.6 and 4.8, bovine serum albumin and ovalbumin, with a load of 220 mg each. The other test was the isolation of 10 mg of human growth hormone isohormone B from a mixture of closely migrating other isohormones. The three-step procedure comprises of: (1) separation into zones of homoge neous protein by gel electrofocusing; (2) excision of the zones of homogeneous protein from the gel followed by concentration of the protein to a small volume of solution by means of Steady-State Stacking; (3) purification from polyacrylamide-like contaminants and non-volatile buffers by gel filtration followed by lyophilization. The average overall recovery was 70--80%. Key words: protein; isolation method; electrofocusing; Steady-State Stacking.

INTRODUCTION A m o n g t h e v a r i o u s a p p r o a c h e s t o p r e p a r a t i v e gel e l e c t r o p h o r e s i s [ 1 ] a n d electrofocusing [2], the original, rudimentary preparative method was zone e x c i s i o n f r o m gels a f t e r s e p a r a t i o n b y o n e o f t h e gel e l e c t r o p h o r e t i c m e t h o d s , f o l l o w e d b y s o m e f o r m o f p r o t e i n r e c o v e r y f r o m t h e slices. T h i s procedure offered several advantages over the more sophisticated other methods: first, the possibility to recover multiple protein zones simultan e o u s l y in h o m o g e n e o u s f o r m ; s e c o n d , t h e a b s e n c e o f a n y risk in l o s i n g t h e p r o t e i n in t h e e l u t i o n c h a m b e r , o r b y i l l - c h o s e n e l e c t r o p h o r e t i c o r e l u t i o n conditions; third, the economic advantage of requiring only extremely simple apparatus. T h i s is n o t a n o r i g i n a l o b s e r v a t i o n . L e w i s a n d C l a r k [ 3 ] a n d S u l i t z e a n u e t

Abbreviations: EF, gel electrofocusing; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; hGH, human growth hormone; MZE, multiphasic zone electro. phoresis :e Steady-State Stacking.

172 al. [4] have applied this simple approach to preparative gel electrophoresis, without, however, providing a comprehensive, widely applicable m e t h o d for protein isolation in high yield. The same concept of protein preparation also underlies the methods of Bont et al. [5] and Dudman and Zerner [6], but their procedures already rely on the availability of relatively complex apparatus. To establish a single, comprehensive and widely applicable procedure leading to the isolation of homogeneous proteins in the amounts required by present routine chemical and physico-chemical methods of protein analysis, viz. ten to several hundred milligrams, three-component methods available in the literature appeared particularly well suited: (1) Separation by gel electrofocusing (EF), in view of its load capacity which is higher than that of polyacrylamide gel electrophoresis (PAGE) by at least one order of magnitude [2]. This method, restricted in principle to the case where the species to be separated differ in net charge b u t not in size, appears far more widely applicable. Thus, the effective use of EF extends to the surprisingly numerous cases in biochemical separations where the procedure of optimization of pore size in PAGE [7] yields a pore size o p t i m u m at '0 %T', i.e. in a 'no-gel'. From the viewpoint of the subsequent procedural steps in protein isolation, EF in simple buffers [8] seems advantageous compared with EF using Ampholine-type carrier constituents. Other advantages of gel electrofocusing, in particular in relation to density-gradient electrofocusing, have previously been discussed [2[. (2) Protein extraction from gel slices and their concentration into a small volume of buffer, using Steady-State Stacking (also referred to as multiphasic zone eiectrophoresis (MZE) [9,10] or isotachophoresis (ITP) [2]). This procedure, and suitable simple apparatus, have recently been elaborated [11] in application to isotopically labeled protein, subjected to concentration in a gel containing 1--3 mg/ml of 'extraneous protein' to avoid adsorption losses to polyacrylamide. Although high recovery in terms of isotope was demonstrated, protein was not physically isolated and checked for purity. Thus, inclusion of this step into a comprehensive isolation procedure required some further work. An alternative procedure [12] seemed less well suited for application to large numbers of gel slices and large sample volumes; it did not take advantage of the procedural comfort of concentration b y Steady-State Stacking. (3) Purification of the product from contaminants derived from polyacrylamide. This procedure, including the required freeing of Sephadex from soluble dextran, has been reported (see Appendix I * to ref. 13) and seemed directly applicable to preparation of the product for lyophilization. This study aimed at combining these three steps into a single, comprehensive procedure for protein isolation, and at proving its efficacy on model * Available from the authors upon request.

173 as well as realistic fractionation problems of a representative degree of difficulty. MATERIALS AND METHODS

1. Proteins Bovine serum albumin (BSA) (No. 2293-01, Reheis), ovalbumin (No. 32467. Calbiochem.) and human growth hormone (hGH) (No. P-6, supplied by the National Pituitary Agency) were used. Buffers: CalTier constituent buffers for EF reported previously [2,14] and listed in Appendix I were used.

2. Electro focusing on polyacrylamide gel The Pyrex apparatus previously described [7] was used with Upper Buffer Reservoirs for 6 and 18 mm i.d. tubes. Polymerization at 0--4°C of 1.7 and 25--30 ml polyacrylamide gel (5 %T, 15 %TDATD[15]) was carried out, for each of the tube sizes, as described previously [7]. Gels contained the buffer mixture listed in Appendix I, at a final gel buffer concentration equalling a quarter that stated in Appendix I, or, when stated under Results, a fourfold concentrate of the same buffer. In application to hGH, the buffer mixture was enriched by asparagine (final concentration 0.035 M) and taurine (0.08 M). The catholyte was 0.2 N KOH, and the anolyte 0.2 N H2SO4. The sample containing the protein(s) (0.10--200 mg of each) at concentrations varying from 0.1 to 1 mg/ml, and 25% sucrose, was applied on the gel surface and overlayered with an equal volume of 10% sucrose, then with the catholyte. EF was performed at 0--4°C at 4 m A / c m 2 until the voltage reached 200 V, and then at a regulated voltage of 200 V. pH gradients across the gel were measured by the automated procedure [16]; on 18 mm gels, the pH positions of the zones were inferred by reference to analytical scale gels of the same height and subjected to identical EF conditions. Gels were either fixed in 12.5% trichloroacetic acid and stained [17], or sliced manually in the position of visible protein zones, or sliced mechanically [18--20]; the wire slicer [20] was adapted (Hoefer Scientific Instruments) to accommodate 18-ram gels. Slices were made alkaline by the addition of 0.05 M Na2HPO4 or NI-I4OH, for a minimal time and preferably at 0°C, and were neutralized.

3. Identification of EF fractions by polyacrylamide gel electrophoresis PAGE analysis of BSA and ovalbumin was carried out at 15 %T, 2 %Cms , in MZE buffer system 2333.0.VII [10] (Appendix II). 0--4°C, using a 3 mm gel slab apparatus (Hoefer Scientific Instruments No. SE-500); combined gel slices and diffusates were applied into alternate slots (2.5 X 18 X 3 mm). In the

t74 case of hGH, analysis was at 12 %T and MZE system 1954.4.0 [10] (Appendix II) was used. The current was regulated at 25 mA/slab (12.5 mA in the case of hGH). The gels were stained [17].

4. Extraction and concentration o f protein from gel slices, using SteadyState Stacking The appm'atus and concentration gel previously described [ 11] were used, e x cep t that collection cups with a dialysis membrane b o t t o m were applied; the dialysis m e m b r a n e was held in place through press-fit by a Kel-F sleeve o f the t y p e described (Fig. 7 of ref. 21). Gel volumes of 35--150 ml were applied, to provide twice or more the volume of the sample. The sample consisted o f tt~e neutralized gel slices and diffusates in 20--30% sucrose, containing B r o mp h e nol Blue. It was overlayered by Upper Buffer. Electrophoresis proceeded at 12 m A / f u n n e l (i.e. per 3.14 cm 2 of gel surface area in the stem) with the collection cup unattached. When the stack reached the stem, the collection cup filled with Upper Buffer was connect ed to the funnel. The current was then decreased to 1--1.2 mA/funnel, and electrophoresis was allowed to proceed for 4--6 h after entrance of the tracking dye into the collection cup and collection of the cup contents; in the case of very lm'ge samples, the second fraction was subjected to electrophoresis overnight. The cup contents were collected for a second time and pooled with the first fraction. The gel was stained [17] to ascertain the absence of protein remaining on the gel.

5. Purification o f the protein concentrate by gel filtration Sephadex G-50 was purified of soluble dextran, and NH4HCO3 buffer, pH 8.0, was prepared as described previously (Appendix I of ref. 13). Columns of 50 X 1.0 or 90 × 2.5 cm (Glenco Scientific No. 3400) were packed with Sephadex G-50 and 0.02 M NH4HCO3 at 0--4°C. T h e y were calibrated, using Dextran Blue and sodium dichromate to mark the void (17o) and internal (Vi) volumes, respectively. The protein concent rat e was applied, allowed to penetrate, and followed by three 3-ml washes. Elution was carried o u t at 0.22--0.30 ml/min, with elution of 10-min fractions. Protein eluate fractions around the Vo were analyzed by ultraviolet spectroscopy (280 nm for BSA and ovalbumin, 277 nm for hGH; Table 5 of ref. 22). The protein containing fractions were pooled and lyophilized.

6. Protein analysis Lyophilizates were weighed and analyzed b y the procedure of L ow ry et al. [23] based on absorbance at 750 nm. Extinction coefficients (1 mg/ml) of 0.770 for BSA, 0.665 for ovaIbumin and 0.695 for hGH [13] were used.

175

7. Analysis of lyophilized product by PAGE PAGE analysis of the lyophilizates was carried out as described in Section 3, except that 6 mm i.d. gel tubes were used [7]. RESULTS

.-

1. Optimization of the duration of EF, at constant load and ionic strength, for the separation between BSA and ovalbumin At a protein load of 8 mg/cm 2 and the ionic strength provided by the gel buffer mixture (Appendix I), BSA and ovalbumin separate on EF ~s two zones, inclusive of theft- various aggregation states, after 70 h at 200 V, 0--4°C (Fig. 1).

2. Maximization of load in relation to the time required for full separation at constant ionic strength When the load of both BSA and ovalbumin was increased from 8 to 80 mg/cm 2 of gel, the time required for full separation increased correspondingly by a factor of 10 (Fig. 2). A load of 8 mg/cm 2 of gel appeared optimal within the practical limits of time available for a routine protein isolation method. c-

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Fig. 1. S e p a r a t i o n o f BSA a n d o v a l b u m i n as a f u n c t i o n o f t h e d u r a t i o n o f EF. Gel b u f f e r : a m i x t u r e o f t h e a m p h o t e r i c b u f f e r s listed in A p p e n d i x I - - final c o n c e n t r a t i o n of e a c h in t h e gel was 0 . 0 1 2 5 M . Gel c o n c e n t r a t i o n : 5 %T, 15 %CDATD. A n o l y t e : 0 . 2 N H2SO4. C a t h o l y t e : 0.2 N KOH. L o a d : 30 m g o f e a c h p r o t e i n / 3 . 1 4 c m 2 gel surface area. B, b o v i n e s e r u m a l b u m i n ; O, o v a l b u m i n . T e m p e r a t u r e : 0--4°C. C u r r e n t : 9.5 m A / 1 8 - m m d i a m e t e r gels u n t i l t h e voltage r e a c h e d 200 V, f o l l o w e d b y 2 3 - - 7 7 h at a regulated p o t e n t i a l of 200 V. 0 a n d 1 d e s i g n a t e d t h e t o p a n d b o t t o m o f t h e E F gel.

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Fig. 2. S e p a r a t i o n o f BSA and o v a l b u m i n in E F as a f u n c t i o n o f p r o t e i n load: The durat i o n o f E F varied f r o m 24 to 249 h in p r o p o r t i o n to p r o t e i n load. O t h e r c o n d i t i o n s a n d n o m e n c l a t u r e as in the legend to Fig. 1.

3. Optimization of ionic strength o f the gel at constant protein load S e p a r a t i o n o f BSA and o v a l b u m i n , at 8 m g / c m 2 each, was carried o u t at f o u r times the c o n c e n t r a t i o n o f the gel b u f f e r m i x t u r e ( A p p e n d i x I). Results were c o m p a r e d with those o b t a i n e d with the u n c o n c e n t r a t e d b u f f e r (Fig. 3). T h e c o n c e n t r a t i o n increase resulted in a p r o p o r t i o n a l , f o u r f o l d increase in t h e d u r a t i o n o f EF. Since this p r o l o n g a t i o n o f E F s e e m e d impractical and n o i m p r o v e m e n t in r e s o l u t i o n was achieved b y an increase in the ionic strength, t h e gel b u f f e r c o n c e n t r a t i o n stated in A p p e n d i x I was p r e f e r r e d .

4. Performance test for the concentration device Since the c o n c e n t r a t i o n device was m o d i f i e d b y a new c o l l e c t i o n cup (see Materials and M e t h o d s ) , an e x p e r i m e n t a l c o n f i r m a t i o n o f t h e previous r e c o v e r y data [11] was required. A t a load o f 100 mg BSA and a gel v o l u m e o f 70 ml, average r e c o v e r y in the collection cup based on L o w r y analysis was 89%.

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5. Isolation o f homogeneous BSA and ovalbumin by separation through EF, concentration through Steady-State Stacking and purification by gel filtration Mixtures o f B S A ( 2 4 0 mg) and ovalbumin ( 2 4 0 mg) were applied on a total of twelve 1 8 - m m diameter gels (40 mg/gel) and separated by EF s i m u l t a n e o u s l y in t w o apparati. Separation was c o m p l e t e after 2 4 - - 3 0 h. A replicate gel was used to locate the separated zones: o n e longitudinal half of it was stained, whilst t h e other was sliced and re-analyzed by PAGE (Fig. 4). Gel sections corresponding to the separated zones were t h e n excised and applied to c o n c e n t r a t i o n gels o f 5 0 - - 1 5 0 ml. Recoveries after c o n c e n t r a t i o n , purification and l y o p h i l i z a t i o n ranged from 58 to 78% (Table 1). The purity o f t h e p r o d u c t ranged from 89 t o 90% (i.e. 0 . 8 - - 0 . 9 mg L o w r y p r o t e i n / m g weight). The h o m o g e n e i t y o f t h e products was ascertained by PAGE analysis (Fig. 5).

6. Isolation of hGH isohormone B by EF, Steady-State Stacking and gel filtration 2 0 mg of hGH, consisting of i s o h o r m o n e s B (60%), C and D as well as some m i n o r protein c o n t a m i n a n t s , were subjected to EF o n o n e 1 8 - m m i.d.

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179 TABLE 1 BSA AND OVALBUMIN ISOLATION FROM A MIXTURE OF THE TWO PROTEINS, USING SEPARATION BY GEL ELECTROFOCUSING, CONCENTRATION BY STEADY-STATE STACKING AND PURIFICATION BY GEL FILTRATION: OVERALL RECOVERY Load (mg)

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a Electrofocusing, Steady-State Stacking, gel filtration, lyophilization. b MZE buffer system 2860.0. c MZE buffer system 2950.0. d 0.05 M NH4OH. gel. A r e p l i c a t e a n a l y t i c a l scale gel was a n a l y z e d f o r p H g r a d i e n t a n d stained (Fig. 6), t o serve as a guide f o r t h e l o c a t i o n o f z o n e s o n t h e basis of R r a n d excision o f t h e h G H - B c o n t a i n i n g gel slice. F o r e x t r a c t i o n a n d c o n c e n t r a t i o n , a S t e a d y ~ S t a t e S t a c k i n g gel o f 35 m l v o l u m e was used. T h e r e c o v e r y of h G H - B a f t e r gel f i l t r a t i o n , a n d l y o p h i l i z a t i o n , based on L o w r y analysis, was 77.6%. T h e p u r i t y o f t h e p r o d u c t ( m g L o w r y p r o t e i n / m g weight) was 7 0 - 78%. T h e h o m o g e n e i t y o f t h e p r o d u c t , i s o h o r m o n e B o f h G H , is d e m o n s t r a t e d b y re-analysis o f t h e l y o p h i l i z e d p r o d u c t o n P A G E (Fig. 7). DISCUSSION A p p l i c a t i o n t o t h e d e m a n d i n g m o d e l f r a c t i o n a t i o n s y s t e m o f BSA a n d o v a l b u m i n o f t h e c o m p r e h e n s i v e p r o t e i n isolation m e t h o d consisting o f s e p a r a t i o n b y E F , c o n c e n t r a t i o n b y S t e a d y - S t a t e S t a c k i n g and p u r i f i c a t i o n b y gel f i l t r a t i o n has s h o w n t h a t t h e p r o c e d u r e is c a p a b l e o f resolving loads o f several h u n d r e d milligrams o f p r o t e i n in high yield (Table 1) w i t h i n a p e r i o d o f less t h a n o n e w o r k w e e k f o r a single investigator. T h e p r o c e d u r e r e q u i r e s v e r y little specialized e q u i p m e n t : (a) U p p e r B u f f e r reservoirs for 18 m m i.d. gels ( t h e m a x i m u m t o l e r a b l e w i t h i n t h e h e a t d i s s i p a t i o n c h a r a c t e r istics o f glass a p p a r a t u s a n d c o m p a t i b l e w i t h a r e a s o n a b l e r a t e o f electrophoresis [ 7 , 1 ] ) w h i c h can b e f i t t e d i n t o n e a r l y all of the available a p p a r a t u s t y p e s f o r a n a l y t i c a l P A G E - - a p p a r a t u s f o r 1 5 - m m i.e. t u b e s is c o m m e r cially available; (b) t h e c o n c e n t r a t i o n gel a p p a r a t u s [11] w h i c h can e i t h e r

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:~-"; i ' , / ' : ' ~ : ~ ~, ~

~1~

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(*

,' , I ; ~ . , z # ' , . /

:

v

~

: [.~ • ; ; ~ ~ ~ ~ ~ -

~,=, ~

~.,.~:

•

.

~::~

'

-

/

tI~ ;

:

~ ~! _!

,

.':.'+~;~:

.17.: ~

~

,

. . ........

:

~~

,.

,~

' ~ :

J:

:,~ -(:~

:

' ~ " . " ~:; I ~

.~. ~ . ~ _ L : ~ : ~ ; I ~ . ¢ ~ ~_

.'~'~

~ ' 1 ~

~ ~ - : . , : ~._~ _' ,.~ ~ . ~ ; ~ j

~. _ .o ~ . , ~ ,

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:

Fig. 6. Preparative s e p a r a t i o n o f i s o h o r m o n e s o f h u m a n g r o w t h h o r m o n e by EF. A mixture o f h G H i s o h o r m o n e s was s e p a r a t e d by E F u n d e r the c o n d i t i o n s described in the legend to Fig. 4 e x c e p t for: gel b u f f e r (0.035 M asparagine and 0.080 M taurine (final c o n c e n t r a t i o n s ) were a d d e d ) ; Load (21.5 mg o f h G H ) ; the resulting p H gradient ( t o p ) and p r o t e i n p a t t e r n ( b o t t o m ) are s h o w n ; t h e d o t t e d line d e n o t e s the p o s i t i o n o f the pl o f hGH-B; p r o t e i n c o n t a i n e d in slices and diffusates were allowed to c o n c e n t r a t e on a Stacking Gel (MZE b u f f e r s y s t e m 1954.4, operative at pH 7.24) at a sample/gel volume ratio o f 0 . 3 5 ; c u r r e n t of 4 m A / c m 2 for 3 h f o l l o w e d by 0.4 m A / c m 2 for 17 h. Fig. 5. I d e n t i f i c a t i o n o f the p r o d u c t s o f preparative E F as s e p a r a t e d BSA and ovalbumin. E F was carried o u t o n 11 gels as s t a t e d in t h e legend to Fig. 4. The total load o f each protein per 11 gels o f 18 m m d i a m e t e r was 220 mg. A f t e r EF, slices were allowed to diffuse at pH 10.5 for 3--4 h. The p r o t e i n in slices and diffusates was c o n c e n t r a t e d by SteadyState Stacking using a gel o f 5 %T, 15 %CDATD (MZE s y s t e m 2950, operative at pH 10.45) at a sample/gel v o l u m e ratio o f 0.4; C u r r e n t ; 4 m A / c m 2 for 24 h f o l l o w e d by 0.4 mA/cm:: for 36 h. The c o m b i n e d c o l l e c t i o n cup c o n t e n t s (2 x 1 ml) were purified by gel filtration ( S e p h a d e x G-50, 1 x 60 cm and 2.5 x 90 cm, 0.02 M NH4HCO 3. pH 8.0), l y o p h i l i z e d , redissolved and a n a l y z e d by P A G E (MZE s y s t e m 2333.0.VII, 15 %T, 2%CBis, O°C) as s h o w n .

182

ORIGINAL

ISOLATED (pl = 5,20)

Rf , "

.

:

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-0

",..:_, ..'.. '

Rf

>i :(:i:i :•:};
'

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Fig. 7. I d e n t i f i c a t i o n o f t h e i s o l a t e d h G H . T h e l y o p h i l ] z e d m a t e r i a l d e r i v e d f r o m t h e e x p e r i m e n t d e s c r i b e d in t h e l e g e n d t o Fig. 6 w a s r e d i s s o l v e d a n d a n a l y z e d b y P A G E ( M Z E system 1954.4.0, 12%T, 2 %Csis, 0°C).

183 easily be constructed or improvised by a t t a c h m e n t of a dialysis bag to the b o t t o m of a gel tube [21]. The limitation of the concentration step (Steady-State Stacking) to 4 m A / c m 2, even in case of very stable proteins like ovalbumin and BSA, implies that one needs to devote a full workday, or more, to this step. This is presumably due to the considerable heating within stacks containing large amounts of protein [31]. Prior to electrophoretic concentration it appem~s necessary to solubilize isoelectrically condensed zones by short exposure to high (or presumably low) pit at low temperature, unless an operative pH of 10.5 (MZE system 2950; Table 1) is applied during the concentration step. At p H 9 . 5 (0°C) Stacking Gels failed to extract ovalbumin efficiently (Table 1). After alkalinization, concentration gels at any pH [10] can presumably be applied. Alkalinization would not be expected to be necessary when preparative PAGE is substituted for preparative EF as the fractionation method. Possibly, detergent solubilization may be applicable in lieu of alkaline solubilization; it has been shown to be compatible with maintenance of at least some biological and enzymatic activities and conformational stability [24--27]. The isolation of homogeneous proteins by excision from gel slices is limiteci b~ the degree of distribution overlap between adjacent zones, and by the capability to slice between non-overlapping protein distributions. Thus, the demonstrated three-step method works, with the yields and degrees of homogeneity shown in this study, only when distribution overlap has been abolished through adequate separation of the components of interest from their neighboring zones. In electrofocusing, such resolution is aided by pH gradient flattening [28]. In each case, it depends on the relative concentrations of neighboring zones, and on their numbers, which again depend on the degree of prefractionation apphed to the problem. As a general isolation strategy, it frequently appears necessary to concentrate on a single component, to resolve the protein of interest clearly and widely from its neighboring zones m~d to select a starting material enriched as much as:possible in the c o m p o n e n t of interest before attempting isolation b y the suggested procedure or any other. Thus, the illustration of hGH-B isolation applies only to a properly selected starting mixture in which the c o m p o n e n t of interest is dominant; for isolation of other hGH isohormones, one would have to prepare (in this case by partial enzymatic digestion) a starting mixture enriched in the other desired isohormone species. Nonetheless, there must exist m a n y cases, in which a single pass through any series of isolation steps would fail to provide full separation, requiring a second or even third refractionation, by the proposed or any other method. Also, it is probably rare to find fractionation problems in which a large number of zones is resolved w i t h o u t significant distribution overlap across neighboring zones, making it possible to apply the proposed m e t h o d to numerous zones simultaneously. Still, cases of multiple zones well resolved from each other

184 exist; the proposed method lends itself to such cases of simultaneous isolations, in contrast to the consecutive elution techniques of preparative PAGE [1]. Thus, the sample fractionations, by which we have attempted to validate the method while yielding a measure of relative effectiveness and load capacity, cannot possibly sel~ce as realistic models for every kind of protein fractionation problem. This does not in any way diminish the claim that the proposed procedure is simple and non-hazardous and tolerates high load, as compared with other presently available protein isolation methods. From the instrumental viewpoint, the isolation procedure still needs to be developed further: for simultaneous isolation of more than three zones, a concentration gel apparatus comprising of more than three gel funnels is needed. A prototype with 10 funnels has been constructed and tested (Fakunding, C., Nguyen, N.Y., DeFonzo, J. and Chrambach, A., in preparation). Smaller-sized gel columns than those used in the present apparatus (which was originally constructed for a single elution peak divided into ascending, center and descending pools and for the relatively large eluate volumes of preparative elution PAGE) would be required in application to the concentration of smaller amounts of protein than used in this study, in view of the need for saturating the non-specific adsorption sites of polyacrylamide for protein if one is to attain an adequate yield [29]. For the preparation of stained guidestrips, a longitudinal slicer [30] modified for application to 18 mm i.d. cylindrical gels and equipped with an asymmetrically positioned cutting wire would be desirable, to provide a narrow guidestrip in lieu of a separate gel run in parallel. Furthermore, the multiple cylindrical gels needed for loads in the range of hundreds of milligrams, as used in this study, should be replaced by one or two gel slabs of the same cross-section. Such preparative slabs should also have an advantage in the rate of heat dissipation, in view of their large surface to mass ratios as compared to gel cylinders. For application of such slabs as concentration gels, slab-slicing both in the direction of migration and at a right angle to it will be required, as well as a mechanical mode of correcting the positions of zones on the stained guidestrip to the corresponding positions of zones on the unstained gel. Instrumental developments in all of these directions are in progress. In summary, a generally applicable protein isolation method is described which is applicable to the range of ten to several hundred milligrams per single component. Its overall recovery is in the order of 70%. It involves protein separation by gel electrofocusing (potentially also gel electrophoresis), followed by excision of get slices, protein concentration by Steady-State Stacking, buffer exchange and purification by gel chromatography, followed by lyophilization. The method excels in procedural and instrumental simplicity, and should prove useful in providing homogeneous proteins in much larger number and a m o u n t than presently available for the determination of specific activities, chemical and physico-chemical analysis.

185 S I M P L I F I E D D E S C R I P T I O N O F T H E M E T H O D A N D ITS A P P L I C A T I O N S A p r o t e i n isolation m e t h o d c o n s i s t i n g of s e p a r a t i o n b y gel e l e c t r o f o c u s i n g , e x t r a c t i o n a n d c o n c e n t r a t i o n o f f r a c t i o n s b y S t e a d y - S t a t e Stacking, p u r i f i c a t i o n o f t h e c o n c e n t r a t e b y gel f i l t r a t i o n , a n d l y o p h i l i z a t i o n yields an overall r e c o v e r y o f 7 0 - - 8 0 % w h e n applied t o a 1 : 1 m i x t u r e o f b o v i n e s e r u m a l b u m i n ( p l 4.8) a n d o v a l b u m i n ( p l 4.6) or t o t h e isola. tion of human growth hormone isohormone B from a mixture with other minor growth h o r m o n e i s o h o r m o n e s . T h e load range used was 1 0 - - 2 2 0 m g o f each p r o t e i n .

APPENDIX 1 pH25oc = 4.3 ; K 25OC

=

] 500.

Weight ( g ) / 1 0 0 ml

Final c o n c e n t r a t i o n (M)

Constituent a MES ACES TES Tricine Bicine Glycylglycine Aspargine Taurine Glycine GABA

Tris 3.92 3.92 4.32 2.64 4.96 3.44 3.36 3.40 2.68 2.44

0.4 x 1.2X 1.2 X 1.6x 0.4 × 2.4 x 1.6 x 0.8 x 0.8 x 1.6 ×

10 -l 10 -2 10 -2 10 -2 10 -3 10 -~ 10 -4 10 -4 10 -~ l 0 -~

Constituent

Tris

0.050 0.054 0.047 0.037 0.076 0.065 0.056 0.068 0.089 0.059

0.057X 0.022X 0.092× 0.035× 0.086 X 0.047 x 0.035 x 0.022 × 0.017 X 0.037 X

10 -2 10 -2 10 -3 10 -3 10 -3 10 -3 10 -3 10 -3 10 -3 10 -4

a C o n s t i t u e n t s are d e f i n e d in ref. 8.

APPENDIX 2 see p. 186.

REFERENCES 1 C h r a m b a c h , A. a n d N g u y e n , N.Y. ( 1 9 7 8 ) in E l e c t r o k i n e t i c S e p a r a t i o n M e t h o d s ( R i g h e t t i , P.J., van Oss, C.J. a n d V a n d e r h o f f , J.W., eds.), pp. 3 3 7 - - 3 6 8 . Elsevier] N o r t h - H o l l a n d Biomedical Press, A m s t e r d a m * 2 C h r a m b a c h , A. a n d N g u y e n , N.Y. ( 1 9 7 7 ) in Proc. I n t e r n a t i o n a l W o r k s h o p o n T e c h n o l o g y for P r o t e i n S e p a r a t i o n a n d I m p r o v e m e n t o f B l o o d Plasma F r a c t i o n a t i o n , ( S a n d b e r g , H., ed.), pp. 4 8 4 - - 5 0 7 . R e s t o n , Va. * 3 Lewis, U.J. a n d Clark M.O. ( 1 9 6 3 ) Anal. B i o c h e m . 6 , 3 0 3 - - 3 1 5 4 S u l i t z e a n u , D., Slavin, M. a n d Yecheskeli, E. ( 1 9 6 7 ) Anal. B i o c h e m . 21, 5 7 - - 6 7 5 B o n t , W.S., Geels, J. a n d R e z e l m a n , G. ( 1 9 6 9 ) Anal. B i o c h e m . 27, 9 9 - - 1 0 7 6 D u d m a n , N.P.R. a n d Zerner, B. ( 1 9 7 3 ) Anal. B i o c h e m . 57, 1 4 - - 2 6 7 C h r a m b a c h , A., Jovin, T.M., S v e n d s e n , P.J. a n d R o d b a r d , D. ( 1 9 7 6 ) in M e t h o d s of

* Available f r o m t h e a u t h o r s u p o n r e q u e s t .

K

pH

pH g

pH K

pH Kc

25°C

1 N HC1 HEM 6.82 8616

l N HCI BLstris 5.83 743]

39.44 ml 9.54 g

36.40 ml 9.23 g

1 N H2SO 4 Ammediol 7.22 4279

1 N H3PO4 b Tris 6.89 212'7

1 N H3PO 4 b HEM 5.27 1898

Acetic acid Bistris 5.43 2304

1.25 g 2.77 g

21.34 ml 2.30 g

20.24 ml 1.92 g

28.70 ml 1.97 g

GABA Ammediol 9.68 263

Glycine Tris 8.89 287

Bicine HEM 7.60 385

TES Bistris 6.78 305

Upper buffer

Resolving gel

Stacking gel

Components/1

C o m P o n e n t s / 1 0 0 ml

c t~-1/cm.

a A table listing s o m e o f the p r o p e r t i e s o f t h e s e b u f f e r s y s t e m s is available u p o n request. b 1N=0.5M.

2950.0

2860.0

2330 .VII

1954.4.0

MZE buffer system a

APPENDIX 2

4.12 g 4.34 g

3.00 g 1t.56 g

5.53 g 5.27 g

9.16 g 4.41 g

I N HCI Ammediol 8.23 4417

] N HCI Tris 7.47 44]7

] N HC1 HEM 6.29 4567

1 N HCI Bistris 5.90 4244

Lower buffer

50.00 ml 6.57 g

50.00 ml '7.57 g

50.00 ml 8.20 g

50.00 ml 13.08 g

187

8 9 10

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Protein Separation (Catsimpoolas, N., ed.), Vol. 2, pp. 27--161. Plenum Press, New York * Nguyen, N.Y. and Chrambach, A. (1976) Anal. Biochem. 74,145--153 Jovin, T.M. (1973) Biochemistry 12,871--898 Jovin, T.M., Dante, M.L. and Chrambach, A. (1970) Multiphasic Buffer Systems Output, PE Nos. 196085--196091,259309--259312, 203016. National Technical Information Service, Springfield, VA 22151 Wachslicht, H. and Chrambach, A. (1978) 84 533--538 Karsnas, P. and Roos, P. (1977) Anal. Biochem. 77, 168--175 Yadley, R.A., Rodbard, D. and Chrambach, A. (1973) Endocrinology 9 3 , 8 6 6 - - 8 7 3 Nguyen, N.Y. and Rodbard, D., Svendsen, P.J. and Chrambach, A. (1977) Anal. Biochem. 77, 39--55 Baumann, G. and Chrambach, A. (1976) Anal. Biochem. 70, 32--38 Chidakel, B.E., Nguyen, N.Y. and Chrambach, A. (1977) Anal. Biochem. 77, 216-225 Diezel, W., Kopperschlaeger, G. and Hofmann, E. (1972) Anal. Biochem. 48, 617-620 Peterson, d.I., Tipton, H.W. and Chrambach, A. (1974) Anal. Biochem. 62, 274--280 Tipton, tI., Rumen, N.M. and Chrambach, A. (1975) Anal. Biochem. 69, 323--326 Chrambach, A. (1966) Anal. Biochem. 1 5 , 5 4 4 - - 5 4 8 McCormick, A.G., Wachslicht, H. and Chrambach, A. (1978) Anal. Biochem. 85, 209--218 Li, C.H. (1972) Proc. Am. Philosoph. Soc. 116, 365--382 Lowry, O.H., Rosebrough, N.J., Farr A.L. and Randall, R.J. (1951) J. Biol. Chem. 193,265--275 Newby, A.C., Rodbell, M. and Chrambach, A. (1978) Arch. Biochem. Biophys. 190, 109--117 Newby, A.C. and Chrambach, A. (1978) Biochem. J. 177,623--630 Newby, A.C., Matthews, G. and Chrambach, A. (1978) Anal. Biochem. 9 1 , 4 7 3 - - 4 8 0 Hjelmeland, L.M., Nebert, D.W. and Chrambach, A. (1978) in Electrophoresis (Catsimpoolas, N., ed.) pp. 29--56. Elsevier/North-Holland Publ. Co., Amsterdam * Chrambach, A. and Nguyen, N.Y. (1978) in Electrophoresis (Catsimpoolas, N., ed.), pp. 3--18, Elsevier/North-Holland Publ. Co. Amsterdam * Kapadia, G. and Chrambach, A. (1972) Anal. Biochem. 48, 90--102 Kohler, P.O., Bridson, W.E. and Chrambach, A. (1971) J. Clin. Endocrinol. 32, 70-76 Baumann, G. and Chrambach, A. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 732--736

* Available from the authors upon request.