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Mechanism of binding of mouse interferon to controlled pore glass
15
Biochimica et Biophysica A cta, 580 ( 1 9 7 9 ) 15--23 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 3 8 2 7 0
MECHANISM OF BINDING OF MOUSE I N T E R F E R O N TO C O N T R O L L E D PORE GLASS
IRWIN A. B R A U D E *, V I C T O R G. E D Y ** a n d E R I K D E C L E R C Q * * *
Department of Human Biology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven (Belgium) (Received F e b r u a r y 1 4 t h , 1 9 7 9 )
Key words: Interferon binding mechanism; Controlled pore glass; (Mouse)
Summary Many proteins bind to controlled pore glass; they are either acid elutable or alkali elutable. Mouse interferon is an acid-elutable protein. Since poly(L-lysine) and, to some extent, poly(L-arginine) are also eluted from controlled pore glass under acidic conditions, one may postulate that mouse interferon binds to controlled pore glass via some of the protein's e-amino groups (of lysine) and/or guanidinium groups (of arginine) and the beads' silanol (hydroxyl groups). The necessity of lysine in the binding of interferon to controlled pore glass is further substantiated by the fact that citraconylated interferon does n o t bind to controlled pore glass. A requirement for Lewis acid-base interaction between the beads' B:O3 groups and the amide groups of arginine is unlikely in view of the results obtained with the alternative system, ZrOH, which, being devoid of B:O3, did bind interferon. Since a substantial a m o u n t of interferon could be eluted from controlled pore glass with ethylene glycol and high salt, one may assume that some h y d r o p h o b i c i t y is involved in the binding of interferon to controlled pore glass.
Introduction Controlled pore glass was originally utilized as a molecular sieve [1,2]. However, many proteins [3,4] bind to the beads, and when alkali and/or chaotropic buffers are employed, these proteins are selectively eluted from the beads. Mouse L-929 and human fibroblast interferons also adsorb to controlled pore * Present address: Interferon Laboratories, Memorial SloanoKettering Cancer Center, N e w York, N.Y. 10021, U.S.A. ** Present address: Theodor Kocher Institute, Freiestrasse 1, Postfach 99, CH-3000 Bern 9, Switzerland. *** To whom to address all correspondence at the Rega Institute, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
16 glass, but unlike the above proteins, interferon is eluted under acidic conditions [5]. It is this distinction which permits the high degree of purification of some interferons on controlled pore glass. In this report, evidence is provided suggesting that the binding of mouse interferon to controlled pore glass occurs via regions of high lysine (and/or arginine) content, and the hydroxyl groups of the beads. In addition, hydrophobic binding, but of a less specific nature, may also be involved. Materials and Methods
Chemicals. Controlled pore glass was obtained from Electro-Nucleonics (Fairfield, NJ), human plasma protein fraction from the National Blood Transfusion Service (Belgian Red Cross), poly(L-lysine) (Mr 15 000--30 000), poly(L-arginine) (Mr 15 000--50 000) from Sigma Chemical Co. (St. Louis, MO), zirconium tetrachloride (ZrC14) from Merck (Darmstadt, F.R.G.), and citraconic anhydride from Koch-Light (Colnbrook, Bucks, U.K.). Interferons and interferon assays. The methodology for preparing mouse interferon and assaying its activity has been described previously [6]. Controlled pore glass adsorption chromatography. The technique of controlled pore glass adsorption chromatography has been discussed previously [5,6]. In addition, a variety of elution procedures were tested, which are referred to in Results. Citraconylation of interferon. 1 ml of mouse interferon, containing 4.0. l 0 s units of activity and 1.6 mg protein, was dialyzed against 100 mM sodium phosphate buffer, pH 8.0. To this, 3 pl of citraconic anhydride was added, and while vigorously stirring, the pH was maintained at 8.0 with 5 N NAOH, and the mixtures incubated at 23°C for 4 h. The material was then dialyzed against 0.1 sodium phosphate buffer, pH 8.0. Removal of protein-bound citraconyl groups was obtained by lowering the pH of the sample, with 1 M HC1, to 4.0, and incubating at 39°C for 4 h. The pH was then readjusted with 1 N NaOH to 7.2, and the sample was dialyzed against Dulbecco's phosphate-buffered saline containing 0.14 m NaC1, 2.5 mM KC1, 10 mM Na2HPO4, 2 mM KH2PO4, 1 mM CaC12 and 0.5 mM MgC12 • 6H20. Controlled pore glass adsorption chromatography of poly(L-lysine) and poly(L-arginine). 100 pg of poly(L-lysine) or poly(L-arginine), in 1 ml phosphatebuffered saline, was loaded onto a 1 X 3 cm column of controlled pore glass, equilibrated in the same buffer. The column was then washed with phosphatebuffered saline, followed by 10 mM Tris-HC1 buffer, pH 8.9, and 400 mM glycine-HC1 buffer, pH 2.0. Ten 1 ml fractions of each were collected, and prior to spectrophotometrically (at 208 nm) determining the polymer content of each fraction in a Beckman spectrophotometer (Model number 25), samples containing Tris-HC1 and glycine-HC1 were dialyzed against phosphate-buffered saline. ZrOH adsorption chromatography. Zirconium hydroxide (ZrOH) was prepared essentially as described by Kennedy et al. [7]. While stirring, 1.5 g of ZrC14 was slowly added to 10 ml 1 M HC1. The pH was then gradually increased to 7.0 with 2.0 M NaOH. The slower the rate of increase in pH, the greater the grain size of polymer. The grains were then washed with 3 1 saline, followed by 1 1 phosphate-buffered saline.
17 The chromatographic technique was as follows: 10 ml of crude interferon were loaded onto a 0.5 × 5.0 cm column of ZrOH, equilibrated in phosphatebuffered saline. The column was then washed with phosphate-buffered saline, followed by 400 mM glycine-HC1, pH 3.5; and, four 4-ml and one 4-ml fractions were collected. Finally, the packed material was washed with 400 mM glycine-HC1, pH 2.0, and five 1 ml fractions were collected. Results
Elution profile of controlled pore glass-bound proteins in mouse interferon preparation As previously reported [5], interferon bound to controlled pore glass could be eluted with low pH buffers. However, as the data presented in Fig. 1 demonstrates, interferon preparations also contain other controlled pore glass-binding proteins which are eluted under alkaline conditions. Six and three-tenths million units of crude interferon (6.3 • 104 units/ml) were loaded onto a 0.9 × 12 cm column of controlled pore glass, equilibrated in phosphate-buffered saline. Subsequent to washing the column with phosphate-buffered saline, 10 mM solutions of Tris-HC1, pH 8.0 and 8.5, and glycine-HC1, pH 2.5, the interferon, 4 • 106 units (or 64% o f the starting material) was eluted with 100 mM KC1/ HC1, pH 2.0. From the total protein recovered, 93%, 6.9%, and 0.3% were found in the fractions containing phosphate-buffered saline; 10 mM Tris-HC1,
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Fig. 1 . C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g z a p h y o f i n t e r f e r o n w i t h alkali and acid e l u a n t . I 0 0 m l o f crude i n t e r f e r o n w e r e l o a d e d o n t o a 0 . 9 X 1 2 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w rate w a s 1 0 m l / h , and 6 . 7 m l / f z a e t i o n w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E l , 1 0 m M Tris-HCl, p H 8 . 0 - - 8 . 5 , as E 2 , 1 0 m M g l y c i n e - H C l , p H 2 . 5 , as E 3 , and 1 0 0 m M KCI/HCI, pH 2 . 0 , as E 4 . T h e e f f l u e n t w a s c o n t i n u o u s l y m o n i t o r e d at 2 8 0 n m (o . . . . . -0) a n d t h e f r a c t i o n s w e r e a s s a y e d for i n t e r f e r o n ( e e).
18 pH 8.0--8.5; and 100 mM KC1/HC1, pH 2.0, respectively. Thus, a clear separation of controlled pore glass-bound proteins could be made depending upon variations in pH.
Elution profile of controlled pore glass-bound proteins with a hydrophobic eluant Since many hydrophobic compounds readily adsorb to SiO2 groups [8], the phosphate-buffered saline containing ethylene glycol and NaC1 was tested as an eluant. As indicated in Fig. 2, when 8.0 • l 0 s units (1.6 • l 0 s units/ml) crude interferon were loaded onto a 0.5 × 5.0 cm column of controlled pore glass, 6.2 • 103 units (or 0.78% of the starting material) were eluted unbound, 2.2 • 10 s units (or 28%) were eluted with phosphate-buffered saline containing 10 M ethylene glycol and 1 M NaC1, pH 7.2; and 1 . 0 . 1 0 3 units (or 0.13%) were eluted with 400 mM glycine-HC1, pH 2.0. No activity was detected from fractions containing 10 mM Tris-HC1, pH 8.9. From the total protein recovered, 93%, 4.2%, 2.2% and 0.13% were found in the fractions containing phosphatebuffered saline, phosphate-buffered saline + ethylene glycol + NaC1, 10 mM Tris-HC1, pH 8.9, and 400 mM glycine-HC1, pH 2.0, respectively. These results
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Fig. 2. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g r a p h y o f i n t e r f e r o n w i t h a h y d r o p h o b i c eluant. 5 m l o f c r u d e i n t e r f e r o n w e r e l o a d e d o n t o a 0 . 5 X 5 . 0 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w rate w a s 1 0 m l / h , and 2 m l / f r a c t i o n , c o n t a i n i n g 0.1 rnl h u m a n p l a s m a p r o t e i n ( 2 2 . 5 m g / r n l ) , w e r e c o l l e c t e d . T h e eluant p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 1 0 M e t h y l e n e g l y c o l and 1 M NaC1, as E 2 , 1 0 m M Tris-HCI, p H 8 . 9 , as E 3 , and 4 0 0 m M g l y c i n e - H C l , pH 2 . 0 , as E 4 . T h e e f f l u e n t w a s c o n t i n u o u s l y m o n i t o r e d at 2 8 0 n m (o . . . . . . o ) and f r a c t i o n s ( E 2 - - E 4 w e r e first d i a l y z e d against p h o s p h a t e - b u f f e r e d saline), w e r e a s s a y e d f o r i n t e r f e r o n
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19 suggest that the h y d r o p h o b i c eluant ethylene glycol + NaC1 will remove most, b u t n o t all, b o u n d acid and alkali-elutable proteins.
Elution profile of citraconylated interferon on controlled pore glass Thus far, the p h e n o m e n o n of protein binding to controlled pore glass has been attributed to the bead's repeating silanol structure [4]. However, more than one binding mechanism must occur since at least two distinct classes of proteins (i.e. acid and alkali elutable) are resolved on controlled pore glass (see Fig. 1). Owing to the net negative charge of the beads under loading conditions [9,10], proteins containing regions of high lysine and/or arginine content seemed likely candidates. The positively charged residues of lysine which are chemically accessible have been reported to become reversibly blocked when proteins are treated with citraconic anhydride [11]. As shown in Fig. 3, interferon treated with this reagent (citraconylated interferon) did n o t bind to controlled pore glass. When 2.5 • l 0 s units of citraconylated interferon were loaded onto a 1.0 × 1.5 cm column of controlled pore glass, 2.1 • 10 s units, or 84% o f the starting material, was detected in the drop-through fractions. Fractions collected from the acid wash contained no detectable activity. The converse was observed when 3.2 • l 0 s units of interferon were loaded, and 1.6 • l 0 s units, or 50% o f the applied material, was detected in the acid wash. Thus, interferon that has been treated with citraconic anhydride, no longer
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Fig. 3. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g r a p h y o f c i t r a c o n y l a t e d i n t e r f e r o n ( C T - M I F ) . A 1.0 × 1.5 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline, w a s l o a d e d w i t h e i t h e z 1 m l c i t r a c o n y l a t e d i n t e r f e r o n (A J ) o r i n t e r f e r o n (o o). T e n 1 m l f r a c t i o n s , c o n t a i n i n g 0.1 m l h u m , a n p l a s m a p r o t e i n ( 2 2 . 5 r n g / m l ) w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , a n d 4 0 0 m M glycine-HC1, p H 2.0, as E 2 .
20 binds to controlled pore glass. This is presumably due to the lysine residues becoming temporarily blocked.
Elution profile o f poly(L-lysine) and poly(L-arginine) on controlled pore glass Further p r o o f for the role of lysine in interferon-controlled pore glass interactions is shown in Fig. 4. When 1.6 absorbance units of poly(L-lysine) were applied to a 1.0 X 3.0 cm column of controlled pore glass, 0.9 unit (or 56% of the starting material) and 0.8 unit (or 50%) were eluted with phosphate-buffered saline and 400 mM glycine-HC1, pH 2.0, respectively. None of the polyamino acid was observed in the 10 mM Tris-HC1, pH 8.9, wash. Thus, similar to interferon, the poly(L-lysine) which did bind to controlled pore glass, was eluted under acidic, and n o t under alkaline, conditions. A different elution profile was obtained with poly(L-arginine). When 3.0 absorbance units were loaded, 0.89 unit (or 30% of the starting material) passed u n b o u n d through the column. Two peaks (0.16 unit or 5.3% and 0.26 unit of 8.7% of the starting material) were detected in the alkali wash, while 1.84 units or 61% of the applied material was found in the acid wash. Elution profile o f mouse interferon on ZrOH In addition to silanol groups, controlled pore glass contains at its surface a significant concentration of boron [9]. Thus, via permeations of B203 groups, the beads may give rise to Lewis acid sites. To determine whether mouse interferon binds to controlled pore glass on the basis of Lewis interactions, another polymer, ZrOH [7], which contains hydroxyl groups but is free of boron, was tested as an alternative binding substrate. As indicated in Fig. 5, when 1.0 • 105 units of crude interferon, (1.0 • 104 units/ml), containing 2.7 mg protein (0.27 mg/ml), or a specific activity of 3.7 • 104 units/mg, were loaded onto a 0.5 X 5.0 cm column of ZrOH, no activity
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Fig. 4. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o r n a t o g r a p h y o f p o l y ( L - l y s i n e ) a n d p o ] y ( L - a r g i n i n e ) . 1 m l o f p o l y ( L - l y s i n e ) ( 1 0 0 /~g, A 2 0 8 = 1.6 a b s o r b a n c e units)~ a n d p o l y ( L - a r g i n i n e ) ( 1 0 0 /~g, A 2 0 8 = 3 . 0 a b s o r b a n c e u n i t s ) , in p h o s p h a t e - b u f f e r e d saline, w e r e l o a d e d o n t o a 1.0 X 3 . 0 c m c o l u m n o f c o n t r o l l e d p o r e glass. T e n 1 m l f r a c t i o n s w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , 1 0 m M Tris-HCl. p H 8.9, as E 2 a n d 4 0 0 m M g l y c i n e - H C l , p H 2.0, as E 3. T h e a b s o r b a n c e o f p o l y ( L - l y s i n e ) (e ~ ) a n d p o l y ( L - a r g i n i n e ) (o . . . . . e) f r a c t i o n s w e r e d e t e r m i n e d at 2 0 8 rim.
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Fig. 5. Z r O H a d s o r p t i o n c h r o m a t o g r a p h y o f i n t e r f e r o n . 10 m l o f c r u d e i n t e r f e r o n w e r e l o a d e d o n t o a 0.5 × 5.0 c m c o l u m n o f Z r O H , e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w r a t e w a s a p p r o x i m a t e l y 1 m l / h . F r a c t i o n 1 c o n t a i n e d 10 m l . f r a c t i o n s 2--6 c o n t a i n e d 4 . 0 r n l / f r a c t i o n a n d f r a c t i o n s 7 - - 1 1 , 1 m l ] f r a c t i o n . T h e e l u a n t p h o s p h a t e - b u f f e r e d ~aline is d e n o t e d as E 1 , 4 0 0 m M glycine-HC1, p H 3.5, as E2, a n d 4 0 0 m M glycine-HC1, p H 2.0, as E 3.
was detected in either the phosphate-buffered saline or 400 mM glycine-HC1, pH 3.5, washes. Fractions obtained with 400 mM glycine-HC1, pH 2.0, contained a total of 1.25 • l 0 s units of activity (or 125% o f the applied material). The peak fraction contained 5 • 104 units of activity and 0.044 mg protein, which corresponds to a specific activity of 1.1 • 106 units/mg and represents a 30-fold purification. Thus, interferon binds to ZrOH with about the same selectivity as it binds to controlled pore glass. Discussion
At least t w o classes of protein are eluted from controlled pore glass (Fig. 1), and their mechanisms o f binding are, most likely, different. The surface o f the beads contains essentially only t w o binding sites, silanol and B203 groups [9]. Both require, for protein binding, the presence of amino groups. If silanol, via its hydroxyl groups, were the responsible ligand at physiological conditions, one could predict a binding between its negative charges [9,10] and the protein's positively charged amino acids. To some extent, this hypothesis was borne o u t by the fact that an increase in negative charge o f the beads (by increasing the pH), did n o t lead to a release of interferon. Mouse interferon can also be eluted from controlled pore glass in the presence o f the h y d r o p h o b i c phosphate-buffered saline containing 10% ethylene glycol and 1 M NaC1 (Fig. 2). The presence o f 1 M salt alone does not desorb interferon (data n o t shown); thus, both ethylene glycol and NaC1 (or ethylene glycol alone) are required. It is not clear, however, whether the buffer affected the protein and/or protein-bead interactions, or whether it directly affected the
22 beads, as polyethylene glycol is known to remove indiscriminately all controlled pore glass-bound proteins [10]. The role of lysine residues in interferon-controlled pore glass interactions was first studied by reversibly blocking the protein's e-amino groups (of lysine) with citraconic anhydride [11]. The citraconylated interferon did not bind to controlled pore glass (Fig. 3). Neither did citraconylated human fibroblast interferon bind to controlled pore glass (Edy, V.G., unpublished observations). Although these observations point to the necessity of lysine residues in the binding of both mouse and human interferon to controlled pore glass, one cannot discount the possibility that the citraconylated interferon, as other citraconylated proteins, assumed a conformation which masked the controlled pore glass-binding sites. Both events are, of course, not mutually exclusive. To substantiate further the role of lysine and to establish the possible role of the other basic amino acid, arginine, in the binding of interferon to controlled pore glass we examined the elution profiles of poly(L-lysine) and poly(L-arginine) (Fig. 4). Similar to interferon, poly(L-lysine) bound to controlled pore glass, and controlled pore glass-bound poly(L-lysine) was eluted under acidic, but not alkaline, conditions. Like citraconylated interferon, citraconylated poly(L-lysine) did not bind to controlled pore glass (data not shown). Unlike interferon, however, half of the poly(L-lysine} material applied to controlled pore glass did not bind. This event is difficult to explain considering the fact that the polymer was characterized by the manufacturer as homogeneous (with molecular weights varying from 15 000 to 30 000, as determined by viscosity), and that all of the material (i.e. applied, drop through or acid wash) had the same spectra, peaking at 208 nm. Except for the two peaks of h o m o p o l y m e r detected during the alkali wash, poly(L-arginine) exhibited the same elution profile as poly(L-lysine). Since some of the material was eluted under alkaline conditions, one might conclude that the binding and elution characteristics for this h o m o p o l y m e r is more complex. More than the presence of lysine and arginine must be required for binding, since all proteins contain these ubiquitous amino acids. Therefore regions o f high basic amino acid content or exposure may be the explanation. This is supported by the fact that mouse interferon is an extremely basic protein, with an isoelectric point (pI) of at least 9.5 [ 12]. The elution conditions employed, and the degree of purification obtained with ZrOH, suggests that the B203 groups, via Lewis acid-base interactions, do not play a major role in interferon-controlled pore glass binding (Fig. 5). In addition, the absence of silicon in ZrOH implies that, for interferon binding to occur, the beads require hydroxyl groups only. In view of the results obtained with citraconylated interferon on the one hand (where only sites containing the e-amino groups of lysine are altered), and with ZrOH on the other hand (which provides no Lewis acid sites), the binding of interferon to controlled pore glass via the amide groups (or Lewis bases) of arginine, asparagine, or glutamine of the interferon molecule can be considered as unlikely. The binding interactions between mouse interferon and controlled pore glass are therefore presumed to occur between some of the protein's e-amino groups o f lysine and/or guanidinium groups of arginine and the hydroxyl groups of
23
the beads. An hydrophobic interaction, possibly via such residues as alanine [13], also plays, perhaps less specific, a role. The elution of mouse interferon could result from either a shift in protein conformation [14] or a change in the beads' charge. Since the pK of the silanol groups is between 6 and 8 [15], the latter seems most likely. Acknowledgments We are grateful for the support and encouragement of Prof. P. De Somer. Our appreciation is also extended to Christiane Callebaut for excellent editorial assistance and to Dr. Z. Zaman for many helpful suggestions and the use of the spectrophotometer. This research was supported by grants from the Belgian F.G.W.O. (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) (Krediet No. 3004.75) and the Geconcerteerde Onderzoeksacties (Conventie No. 76/81IV). The predoctoral training of I.A.B. was supported with funds provided by the Belgian-American Educational Foundation, the Fonds Derde Cyclus (Katholieke Universiteit Leuven), and the Belgian Algemene Spaar- and Lijfrente Kas (A.S.L.K.). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Collins, R.L. and Hailer, W. (1973) Anal. Biochem. 54, 47--53 Haller, W. ( 1 9 6 5 ) Nature 2 0 6 , 6 9 3 - - 6 9 6 Bock, H.G., Skene, P., Fleischex, S., Cassidy, P. and Harshman, S. (1976) Science 191,380--383 Mizutani, T. and Mizutani, A. (1976) J. Chromatogr. 120, 206--210 Edy, V.G., Braude, I.A., De Clercq, E,, Billiau, A. and De Somer, P. (1976) J. Gen. Virol. 33, 517--521 Braude, I.A. and De Clercq, E. (1979) J. Chromatogr. 172, 207--219 Kennedy, J.F., Barker, S.A. and H u m p h r e y s , J.D. (1976) Nature 261,242--244 Flanigen, E.M., Bennett, J.M., Grose, R.W., Cohen, J.P., Patton, R.L., Kirchner, R.M. and Smith, J.V. (1978) Nature 271,512--516 Weetall, H.H. and Fibert, A.M. (1974) Methods Enzymol. 34, 59--71 Operation Instructions for Controlled Pore Glass ( 1 9 7 3 ) ElectToNucleonics, Inc. Habeeb, A.F.S.A. and Atassl, M.Z. (1970) Biochemistry 9, 4939--4944 Kawakita, M., Cabrer, B., Taira, H., Rebello, M., Slattery, E., Weideli, H. and Lengyel, P. (1978) J. Biol. Chem. 253, 598--602 Mizutani, T. and Mizutani, A. (1975) J. Chromatogr. 111,214--216 Jariwalla, R., Grossberg, S.E. and S e d m a k , J.J. (1975) Arch. Virol. 49,261--272 Jacobson, B.S., Cronin, J. and Branton, D. (1978) Biochim. Biophys. Acta 506, 81--96
Biochimica et Biophysica A cta, 580 ( 1 9 7 9 ) 15--23 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 3 8 2 7 0
MECHANISM OF BINDING OF MOUSE I N T E R F E R O N TO C O N T R O L L E D PORE GLASS
IRWIN A. B R A U D E *, V I C T O R G. E D Y ** a n d E R I K D E C L E R C Q * * *
Department of Human Biology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven (Belgium) (Received F e b r u a r y 1 4 t h , 1 9 7 9 )
Key words: Interferon binding mechanism; Controlled pore glass; (Mouse)
Summary Many proteins bind to controlled pore glass; they are either acid elutable or alkali elutable. Mouse interferon is an acid-elutable protein. Since poly(L-lysine) and, to some extent, poly(L-arginine) are also eluted from controlled pore glass under acidic conditions, one may postulate that mouse interferon binds to controlled pore glass via some of the protein's e-amino groups (of lysine) and/or guanidinium groups (of arginine) and the beads' silanol (hydroxyl groups). The necessity of lysine in the binding of interferon to controlled pore glass is further substantiated by the fact that citraconylated interferon does n o t bind to controlled pore glass. A requirement for Lewis acid-base interaction between the beads' B:O3 groups and the amide groups of arginine is unlikely in view of the results obtained with the alternative system, ZrOH, which, being devoid of B:O3, did bind interferon. Since a substantial a m o u n t of interferon could be eluted from controlled pore glass with ethylene glycol and high salt, one may assume that some h y d r o p h o b i c i t y is involved in the binding of interferon to controlled pore glass.
Introduction Controlled pore glass was originally utilized as a molecular sieve [1,2]. However, many proteins [3,4] bind to the beads, and when alkali and/or chaotropic buffers are employed, these proteins are selectively eluted from the beads. Mouse L-929 and human fibroblast interferons also adsorb to controlled pore * Present address: Interferon Laboratories, Memorial SloanoKettering Cancer Center, N e w York, N.Y. 10021, U.S.A. ** Present address: Theodor Kocher Institute, Freiestrasse 1, Postfach 99, CH-3000 Bern 9, Switzerland. *** To whom to address all correspondence at the Rega Institute, Minderbroedersstraat 10, B-3000 Leuven, Belgium.
16 glass, but unlike the above proteins, interferon is eluted under acidic conditions [5]. It is this distinction which permits the high degree of purification of some interferons on controlled pore glass. In this report, evidence is provided suggesting that the binding of mouse interferon to controlled pore glass occurs via regions of high lysine (and/or arginine) content, and the hydroxyl groups of the beads. In addition, hydrophobic binding, but of a less specific nature, may also be involved. Materials and Methods
Chemicals. Controlled pore glass was obtained from Electro-Nucleonics (Fairfield, NJ), human plasma protein fraction from the National Blood Transfusion Service (Belgian Red Cross), poly(L-lysine) (Mr 15 000--30 000), poly(L-arginine) (Mr 15 000--50 000) from Sigma Chemical Co. (St. Louis, MO), zirconium tetrachloride (ZrC14) from Merck (Darmstadt, F.R.G.), and citraconic anhydride from Koch-Light (Colnbrook, Bucks, U.K.). Interferons and interferon assays. The methodology for preparing mouse interferon and assaying its activity has been described previously [6]. Controlled pore glass adsorption chromatography. The technique of controlled pore glass adsorption chromatography has been discussed previously [5,6]. In addition, a variety of elution procedures were tested, which are referred to in Results. Citraconylation of interferon. 1 ml of mouse interferon, containing 4.0. l 0 s units of activity and 1.6 mg protein, was dialyzed against 100 mM sodium phosphate buffer, pH 8.0. To this, 3 pl of citraconic anhydride was added, and while vigorously stirring, the pH was maintained at 8.0 with 5 N NAOH, and the mixtures incubated at 23°C for 4 h. The material was then dialyzed against 0.1 sodium phosphate buffer, pH 8.0. Removal of protein-bound citraconyl groups was obtained by lowering the pH of the sample, with 1 M HC1, to 4.0, and incubating at 39°C for 4 h. The pH was then readjusted with 1 N NaOH to 7.2, and the sample was dialyzed against Dulbecco's phosphate-buffered saline containing 0.14 m NaC1, 2.5 mM KC1, 10 mM Na2HPO4, 2 mM KH2PO4, 1 mM CaC12 and 0.5 mM MgC12 • 6H20. Controlled pore glass adsorption chromatography of poly(L-lysine) and poly(L-arginine). 100 pg of poly(L-lysine) or poly(L-arginine), in 1 ml phosphatebuffered saline, was loaded onto a 1 X 3 cm column of controlled pore glass, equilibrated in the same buffer. The column was then washed with phosphatebuffered saline, followed by 10 mM Tris-HC1 buffer, pH 8.9, and 400 mM glycine-HC1 buffer, pH 2.0. Ten 1 ml fractions of each were collected, and prior to spectrophotometrically (at 208 nm) determining the polymer content of each fraction in a Beckman spectrophotometer (Model number 25), samples containing Tris-HC1 and glycine-HC1 were dialyzed against phosphate-buffered saline. ZrOH adsorption chromatography. Zirconium hydroxide (ZrOH) was prepared essentially as described by Kennedy et al. [7]. While stirring, 1.5 g of ZrC14 was slowly added to 10 ml 1 M HC1. The pH was then gradually increased to 7.0 with 2.0 M NaOH. The slower the rate of increase in pH, the greater the grain size of polymer. The grains were then washed with 3 1 saline, followed by 1 1 phosphate-buffered saline.
17 The chromatographic technique was as follows: 10 ml of crude interferon were loaded onto a 0.5 × 5.0 cm column of ZrOH, equilibrated in phosphatebuffered saline. The column was then washed with phosphate-buffered saline, followed by 400 mM glycine-HC1, pH 3.5; and, four 4-ml and one 4-ml fractions were collected. Finally, the packed material was washed with 400 mM glycine-HC1, pH 2.0, and five 1 ml fractions were collected. Results
Elution profile of controlled pore glass-bound proteins in mouse interferon preparation As previously reported [5], interferon bound to controlled pore glass could be eluted with low pH buffers. However, as the data presented in Fig. 1 demonstrates, interferon preparations also contain other controlled pore glass-binding proteins which are eluted under alkaline conditions. Six and three-tenths million units of crude interferon (6.3 • 104 units/ml) were loaded onto a 0.9 × 12 cm column of controlled pore glass, equilibrated in phosphate-buffered saline. Subsequent to washing the column with phosphate-buffered saline, 10 mM solutions of Tris-HC1, pH 8.0 and 8.5, and glycine-HC1, pH 2.5, the interferon, 4 • 106 units (or 64% o f the starting material) was eluted with 100 mM KC1/ HC1, pH 2.0. From the total protein recovered, 93%, 6.9%, and 0.3% were found in the fractions containing phosphate-buffered saline; 10 mM Tris-HC1,
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Fig. 1 . C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g z a p h y o f i n t e r f e r o n w i t h alkali and acid e l u a n t . I 0 0 m l o f crude i n t e r f e r o n w e r e l o a d e d o n t o a 0 . 9 X 1 2 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w rate w a s 1 0 m l / h , and 6 . 7 m l / f z a e t i o n w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E l , 1 0 m M Tris-HCl, p H 8 . 0 - - 8 . 5 , as E 2 , 1 0 m M g l y c i n e - H C l , p H 2 . 5 , as E 3 , and 1 0 0 m M KCI/HCI, pH 2 . 0 , as E 4 . T h e e f f l u e n t w a s c o n t i n u o u s l y m o n i t o r e d at 2 8 0 n m (o . . . . . -0) a n d t h e f r a c t i o n s w e r e a s s a y e d for i n t e r f e r o n ( e e).
18 pH 8.0--8.5; and 100 mM KC1/HC1, pH 2.0, respectively. Thus, a clear separation of controlled pore glass-bound proteins could be made depending upon variations in pH.
Elution profile of controlled pore glass-bound proteins with a hydrophobic eluant Since many hydrophobic compounds readily adsorb to SiO2 groups [8], the phosphate-buffered saline containing ethylene glycol and NaC1 was tested as an eluant. As indicated in Fig. 2, when 8.0 • l 0 s units (1.6 • l 0 s units/ml) crude interferon were loaded onto a 0.5 × 5.0 cm column of controlled pore glass, 6.2 • 103 units (or 0.78% of the starting material) were eluted unbound, 2.2 • 10 s units (or 28%) were eluted with phosphate-buffered saline containing 10 M ethylene glycol and 1 M NaC1, pH 7.2; and 1 . 0 . 1 0 3 units (or 0.13%) were eluted with 400 mM glycine-HC1, pH 2.0. No activity was detected from fractions containing 10 mM Tris-HC1, pH 8.9. From the total protein recovered, 93%, 4.2%, 2.2% and 0.13% were found in the fractions containing phosphatebuffered saline, phosphate-buffered saline + ethylene glycol + NaC1, 10 mM Tris-HC1, pH 8.9, and 400 mM glycine-HC1, pH 2.0, respectively. These results
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Fig. 2. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g r a p h y o f i n t e r f e r o n w i t h a h y d r o p h o b i c eluant. 5 m l o f c r u d e i n t e r f e r o n w e r e l o a d e d o n t o a 0 . 5 X 5 . 0 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w rate w a s 1 0 m l / h , and 2 m l / f r a c t i o n , c o n t a i n i n g 0.1 rnl h u m a n p l a s m a p r o t e i n ( 2 2 . 5 m g / r n l ) , w e r e c o l l e c t e d . T h e eluant p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , p h o s p h a t e b u f f e r e d saline c o n t a i n i n g 1 0 M e t h y l e n e g l y c o l and 1 M NaC1, as E 2 , 1 0 m M Tris-HCI, p H 8 . 9 , as E 3 , and 4 0 0 m M g l y c i n e - H C l , pH 2 . 0 , as E 4 . T h e e f f l u e n t w a s c o n t i n u o u s l y m o n i t o r e d at 2 8 0 n m (o . . . . . . o ) and f r a c t i o n s ( E 2 - - E 4 w e r e first d i a l y z e d against p h o s p h a t e - b u f f e r e d saline), w e r e a s s a y e d f o r i n t e r f e r o n
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19 suggest that the h y d r o p h o b i c eluant ethylene glycol + NaC1 will remove most, b u t n o t all, b o u n d acid and alkali-elutable proteins.
Elution profile of citraconylated interferon on controlled pore glass Thus far, the p h e n o m e n o n of protein binding to controlled pore glass has been attributed to the bead's repeating silanol structure [4]. However, more than one binding mechanism must occur since at least two distinct classes of proteins (i.e. acid and alkali elutable) are resolved on controlled pore glass (see Fig. 1). Owing to the net negative charge of the beads under loading conditions [9,10], proteins containing regions of high lysine and/or arginine content seemed likely candidates. The positively charged residues of lysine which are chemically accessible have been reported to become reversibly blocked when proteins are treated with citraconic anhydride [11]. As shown in Fig. 3, interferon treated with this reagent (citraconylated interferon) did n o t bind to controlled pore glass. When 2.5 • l 0 s units of citraconylated interferon were loaded onto a 1.0 × 1.5 cm column of controlled pore glass, 2.1 • 10 s units, or 84% o f the starting material, was detected in the drop-through fractions. Fractions collected from the acid wash contained no detectable activity. The converse was observed when 3.2 • l 0 s units of interferon were loaded, and 1.6 • l 0 s units, or 50% o f the applied material, was detected in the acid wash. Thus, interferon that has been treated with citraconic anhydride, no longer
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Fig. 3. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o m a t o g r a p h y o f c i t r a c o n y l a t e d i n t e r f e r o n ( C T - M I F ) . A 1.0 × 1.5 c m c o l u m n o f c o n t r o l l e d p o r e glass, e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline, w a s l o a d e d w i t h e i t h e z 1 m l c i t r a c o n y l a t e d i n t e r f e r o n (A J ) o r i n t e r f e r o n (o o). T e n 1 m l f r a c t i o n s , c o n t a i n i n g 0.1 m l h u m , a n p l a s m a p r o t e i n ( 2 2 . 5 r n g / m l ) w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , a n d 4 0 0 m M glycine-HC1, p H 2.0, as E 2 .
20 binds to controlled pore glass. This is presumably due to the lysine residues becoming temporarily blocked.
Elution profile o f poly(L-lysine) and poly(L-arginine) on controlled pore glass Further p r o o f for the role of lysine in interferon-controlled pore glass interactions is shown in Fig. 4. When 1.6 absorbance units of poly(L-lysine) were applied to a 1.0 X 3.0 cm column of controlled pore glass, 0.9 unit (or 56% of the starting material) and 0.8 unit (or 50%) were eluted with phosphate-buffered saline and 400 mM glycine-HC1, pH 2.0, respectively. None of the polyamino acid was observed in the 10 mM Tris-HC1, pH 8.9, wash. Thus, similar to interferon, the poly(L-lysine) which did bind to controlled pore glass, was eluted under acidic, and n o t under alkaline, conditions. A different elution profile was obtained with poly(L-arginine). When 3.0 absorbance units were loaded, 0.89 unit (or 30% of the starting material) passed u n b o u n d through the column. Two peaks (0.16 unit or 5.3% and 0.26 unit of 8.7% of the starting material) were detected in the alkali wash, while 1.84 units or 61% of the applied material was found in the acid wash. Elution profile o f mouse interferon on ZrOH In addition to silanol groups, controlled pore glass contains at its surface a significant concentration of boron [9]. Thus, via permeations of B203 groups, the beads may give rise to Lewis acid sites. To determine whether mouse interferon binds to controlled pore glass on the basis of Lewis interactions, another polymer, ZrOH [7], which contains hydroxyl groups but is free of boron, was tested as an alternative binding substrate. As indicated in Fig. 5, when 1.0 • 105 units of crude interferon, (1.0 • 104 units/ml), containing 2.7 mg protein (0.27 mg/ml), or a specific activity of 3.7 • 104 units/mg, were loaded onto a 0.5 X 5.0 cm column of ZrOH, no activity
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Fig. 4. C o n t r o l l e d p o r e glass a d s o r p t i o n c h r o r n a t o g r a p h y o f p o l y ( L - l y s i n e ) a n d p o ] y ( L - a r g i n i n e ) . 1 m l o f p o l y ( L - l y s i n e ) ( 1 0 0 /~g, A 2 0 8 = 1.6 a b s o r b a n c e units)~ a n d p o l y ( L - a r g i n i n e ) ( 1 0 0 /~g, A 2 0 8 = 3 . 0 a b s o r b a n c e u n i t s ) , in p h o s p h a t e - b u f f e r e d saline, w e r e l o a d e d o n t o a 1.0 X 3 . 0 c m c o l u m n o f c o n t r o l l e d p o r e glass. T e n 1 m l f r a c t i o n s w e r e c o l l e c t e d . T h e e l u a n t p h o s p h a t e - b u f f e r e d saline is d e n o t e d as E 1 , 1 0 m M Tris-HCl. p H 8.9, as E 2 a n d 4 0 0 m M g l y c i n e - H C l , p H 2.0, as E 3. T h e a b s o r b a n c e o f p o l y ( L - l y s i n e ) (e ~ ) a n d p o l y ( L - a r g i n i n e ) (o . . . . . e) f r a c t i o n s w e r e d e t e r m i n e d at 2 0 8 rim.
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Fig. 5. Z r O H a d s o r p t i o n c h r o m a t o g r a p h y o f i n t e r f e r o n . 10 m l o f c r u d e i n t e r f e r o n w e r e l o a d e d o n t o a 0.5 × 5.0 c m c o l u m n o f Z r O H , e q u i l i b r a t e d in p h o s p h a t e - b u f f e r e d saline. T h e f l o w r a t e w a s a p p r o x i m a t e l y 1 m l / h . F r a c t i o n 1 c o n t a i n e d 10 m l . f r a c t i o n s 2--6 c o n t a i n e d 4 . 0 r n l / f r a c t i o n a n d f r a c t i o n s 7 - - 1 1 , 1 m l ] f r a c t i o n . T h e e l u a n t p h o s p h a t e - b u f f e r e d ~aline is d e n o t e d as E 1 , 4 0 0 m M glycine-HC1, p H 3.5, as E2, a n d 4 0 0 m M glycine-HC1, p H 2.0, as E 3.
was detected in either the phosphate-buffered saline or 400 mM glycine-HC1, pH 3.5, washes. Fractions obtained with 400 mM glycine-HC1, pH 2.0, contained a total of 1.25 • l 0 s units of activity (or 125% o f the applied material). The peak fraction contained 5 • 104 units of activity and 0.044 mg protein, which corresponds to a specific activity of 1.1 • 106 units/mg and represents a 30-fold purification. Thus, interferon binds to ZrOH with about the same selectivity as it binds to controlled pore glass. Discussion
At least t w o classes of protein are eluted from controlled pore glass (Fig. 1), and their mechanisms o f binding are, most likely, different. The surface o f the beads contains essentially only t w o binding sites, silanol and B203 groups [9]. Both require, for protein binding, the presence of amino groups. If silanol, via its hydroxyl groups, were the responsible ligand at physiological conditions, one could predict a binding between its negative charges [9,10] and the protein's positively charged amino acids. To some extent, this hypothesis was borne o u t by the fact that an increase in negative charge o f the beads (by increasing the pH), did n o t lead to a release of interferon. Mouse interferon can also be eluted from controlled pore glass in the presence o f the h y d r o p h o b i c phosphate-buffered saline containing 10% ethylene glycol and 1 M NaC1 (Fig. 2). The presence o f 1 M salt alone does not desorb interferon (data n o t shown); thus, both ethylene glycol and NaC1 (or ethylene glycol alone) are required. It is not clear, however, whether the buffer affected the protein and/or protein-bead interactions, or whether it directly affected the
22 beads, as polyethylene glycol is known to remove indiscriminately all controlled pore glass-bound proteins [10]. The role of lysine residues in interferon-controlled pore glass interactions was first studied by reversibly blocking the protein's e-amino groups (of lysine) with citraconic anhydride [11]. The citraconylated interferon did not bind to controlled pore glass (Fig. 3). Neither did citraconylated human fibroblast interferon bind to controlled pore glass (Edy, V.G., unpublished observations). Although these observations point to the necessity of lysine residues in the binding of both mouse and human interferon to controlled pore glass, one cannot discount the possibility that the citraconylated interferon, as other citraconylated proteins, assumed a conformation which masked the controlled pore glass-binding sites. Both events are, of course, not mutually exclusive. To substantiate further the role of lysine and to establish the possible role of the other basic amino acid, arginine, in the binding of interferon to controlled pore glass we examined the elution profiles of poly(L-lysine) and poly(L-arginine) (Fig. 4). Similar to interferon, poly(L-lysine) bound to controlled pore glass, and controlled pore glass-bound poly(L-lysine) was eluted under acidic, but not alkaline, conditions. Like citraconylated interferon, citraconylated poly(L-lysine) did not bind to controlled pore glass (data not shown). Unlike interferon, however, half of the poly(L-lysine} material applied to controlled pore glass did not bind. This event is difficult to explain considering the fact that the polymer was characterized by the manufacturer as homogeneous (with molecular weights varying from 15 000 to 30 000, as determined by viscosity), and that all of the material (i.e. applied, drop through or acid wash) had the same spectra, peaking at 208 nm. Except for the two peaks of h o m o p o l y m e r detected during the alkali wash, poly(L-arginine) exhibited the same elution profile as poly(L-lysine). Since some of the material was eluted under alkaline conditions, one might conclude that the binding and elution characteristics for this h o m o p o l y m e r is more complex. More than the presence of lysine and arginine must be required for binding, since all proteins contain these ubiquitous amino acids. Therefore regions o f high basic amino acid content or exposure may be the explanation. This is supported by the fact that mouse interferon is an extremely basic protein, with an isoelectric point (pI) of at least 9.5 [ 12]. The elution conditions employed, and the degree of purification obtained with ZrOH, suggests that the B203 groups, via Lewis acid-base interactions, do not play a major role in interferon-controlled pore glass binding (Fig. 5). In addition, the absence of silicon in ZrOH implies that, for interferon binding to occur, the beads require hydroxyl groups only. In view of the results obtained with citraconylated interferon on the one hand (where only sites containing the e-amino groups of lysine are altered), and with ZrOH on the other hand (which provides no Lewis acid sites), the binding of interferon to controlled pore glass via the amide groups (or Lewis bases) of arginine, asparagine, or glutamine of the interferon molecule can be considered as unlikely. The binding interactions between mouse interferon and controlled pore glass are therefore presumed to occur between some of the protein's e-amino groups o f lysine and/or guanidinium groups of arginine and the hydroxyl groups of
23
the beads. An hydrophobic interaction, possibly via such residues as alanine [13], also plays, perhaps less specific, a role. The elution of mouse interferon could result from either a shift in protein conformation [14] or a change in the beads' charge. Since the pK of the silanol groups is between 6 and 8 [15], the latter seems most likely. Acknowledgments We are grateful for the support and encouragement of Prof. P. De Somer. Our appreciation is also extended to Christiane Callebaut for excellent editorial assistance and to Dr. Z. Zaman for many helpful suggestions and the use of the spectrophotometer. This research was supported by grants from the Belgian F.G.W.O. (Fonds voor Geneeskundig Wetenschappelijk Onderzoek) (Krediet No. 3004.75) and the Geconcerteerde Onderzoeksacties (Conventie No. 76/81IV). The predoctoral training of I.A.B. was supported with funds provided by the Belgian-American Educational Foundation, the Fonds Derde Cyclus (Katholieke Universiteit Leuven), and the Belgian Algemene Spaar- and Lijfrente Kas (A.S.L.K.). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Collins, R.L. and Hailer, W. (1973) Anal. Biochem. 54, 47--53 Haller, W. ( 1 9 6 5 ) Nature 2 0 6 , 6 9 3 - - 6 9 6 Bock, H.G., Skene, P., Fleischex, S., Cassidy, P. and Harshman, S. (1976) Science 191,380--383 Mizutani, T. and Mizutani, A. (1976) J. Chromatogr. 120, 206--210 Edy, V.G., Braude, I.A., De Clercq, E,, Billiau, A. and De Somer, P. (1976) J. Gen. Virol. 33, 517--521 Braude, I.A. and De Clercq, E. (1979) J. Chromatogr. 172, 207--219 Kennedy, J.F., Barker, S.A. and H u m p h r e y s , J.D. (1976) Nature 261,242--244 Flanigen, E.M., Bennett, J.M., Grose, R.W., Cohen, J.P., Patton, R.L., Kirchner, R.M. and Smith, J.V. (1978) Nature 271,512--516 Weetall, H.H. and Fibert, A.M. (1974) Methods Enzymol. 34, 59--71 Operation Instructions for Controlled Pore Glass ( 1 9 7 3 ) ElectToNucleonics, Inc. Habeeb, A.F.S.A. and Atassl, M.Z. (1970) Biochemistry 9, 4939--4944 Kawakita, M., Cabrer, B., Taira, H., Rebello, M., Slattery, E., Weideli, H. and Lengyel, P. (1978) J. Biol. Chem. 253, 598--602 Mizutani, T. and Mizutani, A. (1975) J. Chromatogr. 111,214--216 Jariwalla, R., Grossberg, S.E. and S e d m a k , J.J. (1975) Arch. Virol. 49,261--272 Jacobson, B.S., Cronin, J. and Branton, D. (1978) Biochim. Biophys. Acta 506, 81--96