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Biological Applications on Spherogel TSK-SW-TypeGel A New High-Performance Support for Aqueous Size Exclusion Chromatography R. SOMACK, V. S. McKAY, and J. W. GILES Altex Scientific, Inc., 1780 Fourth Street, Berkeley, C A 94710
Among the p r i n c i p a l techniques used f o r the p u r i f i c a t i o n o f proteins and other macromolecules o f b i o l o g i c a l systems are s i z e e x c l u s i o n (gel f i l t r a t i o n ) , ion-exchange, and b i o s p e c i f i c a f f i n i t y chromatography. Several very useful c e l l u l o s i c , dextran, and polyacrylamide column m a t e r i a l s have been a v a i l a b l e f o r some time f o r these types o f chromatography. The e s s e n t i a l p r o p e r t i e s o f these s t a t i o n a r y phase m a t e r i a l s are t h e i r l a r g e p o r o s i t i e s and h y d r o p h i l i c surfaces which minimize undesirable i n t e r a c t i o n s b e tween s o l u t e s and the s t a t i o n a r y phase. While these m a t e r i a l s have been extremely useful i n uncountable s t u d i e s , they are not without disadvantages. T h e i r drawbacks r e s u l t from a lack o f r i g i d i t y which causes t h e i r degree o f s w e l l i n g t o change markedly in response t o changes i n pH o r i o n i c s t r e n g t h . An even more s e r i o u s consequence o f t h e i r n o n - r i g i d i t y i s a high s u s c e p t a b i l i t y t o compression when s o l v e n t flow rates exceed r a t h e r low limits. The r e s t r i c t i o n t o q u i t e slow flow rates when these comp r e s s i b l e gels are used often extends the time r e q u i r e d f o r a t y p i c a l separation t o many hours o r even a few days (1). U n t i l r e c e n t l y , most e f f o r t s t o develop r i g i d s i z e e x c l u s i o n o r ion-exchange supports which do not adsorb or denature p r o t e i n s and which would be compatible with a high performance l i q u i d chromatography have met with l i t t l e s u c c e s s . "Controlled porosity g l a s s " supports have been developed {2, 3, 4 ) , but the r e a c t i v e exposed s i l a n o l groups i r r e v e r s i b l y adsorb o r denature many p r o t e i n s ( 5 ) . Attempts t o e l i m i n a t e these e f f e c t s by c o a t i n g the glass s u r f a c e with various reagents have met with l i m i t e d success ( £ , _7» 8 , £ , 10.). However, masking the r e a c t i v e s i l i c a surface with carbohydrate l i k e bonded phases appears t o have e l i m i n a t e d some o f these e f f e c t s (1J_, 1 2 ) . More r e c e n t l y , maximum coverage m i c r o p a r t i c u l a t e bonded s i l i c a supports have been described which appear t o e x h i b i t the d e s i r e d p r o p e r t i e s o f low adsorption and high r e s o l u t i o n ( 1 3 , 1 4 ) . In t h i s study we have examined the s u i t a b i l i t y o f Spherogel-TSK Type SW , a new s i z e e x c l u s i o n support R
0-8412-0586-8/80/47-138-285$05.00/0 © 1980 American Chemical Society Provder; Size Exclusion Chromatography (GPC) ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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o f the l a t t e r t y p e , f o r r a p i d , high r e s o l u t i o n separations comonl y encountered i n biochemical r e s e a r c h . Experimental Methods High Performance L i q u i d Chromatography. A l l separations were performed using an A l t e x S c i e n t i f i c (1780 Fourth S t r e e t , Berkeley, CA 94710) Model 320 Advance Research Chromatograph, c o n s i s t i n g o f a model 100A dual p i s t o n a n a l y t i c a l pump, a Model 153 UV d e t e c t o r , a Model 210 i n j e c t i o n v a l v e , and a Model 155 recorder. The columns (600 x7.5mm) evaluated were the Spherogel TSK-SW-2000 and SW-3000 ( A l t e x ) . Unless otherwise s t a t e d , a l l separations were c a r r i e d out a t 23° - 25°. Proteins. Standard p r o t e i n s f o r column c a l i b r a t i o n curves were obtained from Boehringer Mannheim. The methods o u t l i n e d by Latham e t a l (15) were followed^to prepare and l a b e l crude r a t l i v e r nuclear e x t r a c t with Π Π -triiodothyronine. Normal con t r o l serum was from Ortho Diagnostics and i n s t a n t non-fat dry milk from C a r n a t i o n . Alpha-chymotrypsin and a l p h a - c a s e i n were purchased from Sigma. Crude myosin subfragment 1 (SI) was p u r i f i e d by alpha-chymotryptic d i g e s t i o n o f myosin using the method o f Weeds and T a y l o r (16>). A f r a c t i o n c o n t a i n i n g a l l three myosin l i g h t chains was i s o l a t e d as described by H o l t and Lowry ( 1 7 ) . A l l myosin derived p r o t e i n s were d i a l i z e d against mobile phase b u f f e r c o n s i s t i n g o f 50 mM Na3P04 (pH 7 . 4 ) , 0 . 2 M (NH4) S04, 1 mM EDTA, 0.2 mM d i t h i o t h r e c t o ! and 0.02% NaN3* %
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Assays, Protein concentrations were measured by the method o f Bradford (18) and the various c o n t r a c t i l e p r o t e i n ATPase a c t i v i t i e s by the method o f Martin and Doty (.19). Gel e l e c t r o phoresis was c a r r i e d out by the method o f Ames (20) on 1.5 mm polyacrylamide slabs using the discontinuous SDS b u f f e r system o f Laemmli (2]_). Dried gels were scanned a t 550 nm f o r densiometry measurements. Results and Discussion C a l i b r a t i o n Curves and E f f i c i e n c y . Protein c a l i b r a t i o n curves f o r both the SW-2000 and SW-3000 columns are shown i n Figure 1. Good l i n e a r i t y was e v i d e n t over the range o f molecular weights used with each column. However, cytochrome C which i s a s m a l l , very b a s i c p r o t e i n e l u t e d sooner than expected when chromatographed on the SW-2000 column. The f r a c t i o n a t i o n o f a mixture o f four standard p r o t e i n s ranging i n molecular weight from 15,000 t o 150,000 on the SW-2000 column i s shown i n the same f i g u r e . The recovery o f absorbance units was e s s e n t i a l l y complete and no a d s o r p t i o n o f the proteins t e s t e d was evident a t the concentration of phosphate b u f f e r used ( 0 . 2 M).
Provder; Size Exclusion Chromatography (GPC) ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Figure 1. Protein calibration curves for the Spherogel TSK-SW 2000 and SW 3000 columns. Ten μΣ containing 10-100 fig of each protein in 0.2M KPO buffer (pH 6.8) was chromatographed in the same buffer at 1.0 mL/min. Detection was at 254 nm X 0.16 AUFS; pressure: 500 psi. A chromatogram of 4 proteins on the SW 2000 column is also shown. f
Provder; Size Exclusion Chromatography (GPC) ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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Figure 2 shows the e f f e c t o f flow r a t e on column e f f i c i e n c y using the SW-2000 column with cytochrome C. The column e f f i c i e n cy expressed as the number o f t h e o r e t i c a l p l a t e s (N) was depend ent on flow r a t e , a r e s u l t t y p i c a l o f s i z e e x c l u s i o n chromato graphy. F r a c t i o n a t i o n o f Serum and Column C a p a c i t y . A Fractiona t i o n o f human serum on the SW-3000 column i s shown i n Figure 3. A d e f i n i t i v e i d e n t i f i c a t i o n o f the p r o t e i n s i n each peak i s not p o s s i b l e , however, the e l u t i o n times o f the peaks a t 13-14 min. and 15 min. are c l o s e t o the times which would be expected f o r gamma-globulins and albumins, two o f the p r i n c i p a l c l a s s e s o f serum p r o t e i n s . These data a l s o i n d i c a t e the l o a d i n g c a p a c i t y o f t h i s column with serum. More than 14 mg. o f u n d i l u t e d serum was i n j e c t e d before evidence o f overloading i n the form o f band broadening and peak d i s t o r t i o n was observed. Milk P r o t e i n s . S i z e e x c l u s i o n chromatography i s commonly used i n biochemistry t o observe i n t e r a c t i v e processes between proteins. We tested the c a p a b i l i t y o f the SW-2000 gel to follow such phenomena i n m i l k . Figure 4 shows the 254 nm absorbing p r o f i l e s obtained a f t e r chromatographing i d e n t i c a l volumes o f three d i l u t i o n s o f non-fat dry milk i n 0.2 M PO4 b u f f e r , a t pH 7. While the data are not amenable t o q u a n t i t a t i v e a n a l y s i s , d i l u t i o n appears t o have caused disaggregation o f higher molecular weight m a t e r i a l i n the f i r s t peak with a concomitant accumulation o f lower molecular weight material i n the t h i r d peak. In order t o e s t a b l i s h t h a t t h i s e f f e c t was not due to the d i f f e r e n c e i n t o t a l mass chromatographed, 10 y l o f u n d i l u t e d whole milk was i n j e c t e d using a 10 y l loop and the r e s u l t compared t o a run where 100 μΐ o f a 1:10 d i l u t i o n was i n j e c t e d using a 100 μΤ l o o p . Although the same sample mass was a p p l i e d i n both c a s e s , the apparent d i s aggregation was s t i l l observed (data not shown). A s i m i l a r e f f e c t was observed i n whole m i l k . Upon rechromatographing m a t e r i a l c o l l e c t e d from the f i r s t peak, a s h i f t o f much o f the material to a l a t e r e l u t i n g p o s i t i o n occurred (Figure 5 ) . A s i m i l a r d i l u t i o n e f f e c t can a l s o be observed by chromatographing a l p h a - c a s e i n alone at d i f f e r e n t concentrations (Figure 6 ) . I n i t i a l l y , the p r o t e i n was chromatographed a t approximately the same concentration a t which i t i s present i n whole m i l k . A f t e r d i l u t i n g the p r o t e i n 1:10, the p r o f i l e c l e a r l y showed the disaggregation o f m a t e r i a l with a molecular weight o f 80,000 daltons o r g r e a t e r . These ob s e r v a t i o n s are c o n s i s t e n t with the known p r o p e r t i e s o f a l p h a c a s e i n , the major p r o t e i n found i n m i l k . Dry milk i s 25% casein by weight, h a l f o f which i s a l p h a - c a s e i n ( 2 2 ) . The a l p h a - c a s e i n f r a c t i o n makes up 50% o f the t o t a l milk p r o t e i n and a l l o f the casein i n milk p a r t i c i p a t e s i n complex, r e v e r s i b l e aggregation (23, 2 4 ) . I t seems reasonable t o speculate t h a t these d i l u t i o n e f f e c t s observed i n whole milk are due t o the d i s a s s o c i a t i o n o f high molecular weight casein aggregates. High performance s i z e
Provder; Size Exclusion Chromatography (GPC) ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
SOMACK ET AL.
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Figure 2. Effect of flow rate on the efficiency (N) of the Spherogel TSK-SW 2000 column. The conditions were as indicated in Figure 1 using cytochrome C as test solute.
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Figure 3. Fractionation of human serum proteins on Spherogel TSKSW 3000. The conditions were as in Figure 1. The analyses were made using (Λ) α 50-μΣ, injection loop with an analyticalflowcell; (B) a 100-L loop with a semipreparative flow cell; or (C, D) a 500-L loop with a preparativeflowcell.
Provder; Size Exclusion Chromatography (GPC) ACS Symposium Series; American Chemical Society: Washington, DC, 1980.
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MW 80,000
