- Email: [email protected]
[8] Interfering and complicating adsorption effects in bioaffinity chromatography
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nium agarose, stirred in an ice bath. Formation of the colored azo gel begins immediately, and coupling is usually complete in 1 0 - 3 0 minutes. During this time, the mixture is allowed to warm to room temperature. If desired, any unreacted diazonium groups '~° may be blocked at this point by addition of a dilute solution of fl-naphthol or of 2,6-di-tert-butylphenol in 5 0 % dimethylformamide. The gel is filtered and washed thoroughly with water, with 50% dimethylformamide, and again with water, and is stored (cold) as a suspension in water or buffer. ~*This procedure is usually unnecessary for small ligands, especially when used in moderate excess in the coupling reaction.
[8] Interfering and Complicating
Adsorption
in Bioaffinity Chromatography
Effects
1
B y P.~DRAIG O'CARRA, STANDISH BARRY, and TADHG GRIFFIN
The term affinity chromatography was originally introduced to denote adsorption chromatography based on enzymatically specific interactions, -~ as distinct from adsorption dependent on gross physicochemical properties, which in this context has generally been referred to as nonspecific adsorption. This terminology is not entirely satisfactory since the term affinity could be validly applied to any form of adsorption, specific or otherwise, while the so-called nonspecific adsorption effects often have quite a high degree of apparent specificity, as evidenced by the enzyme purifications achievable by ion-exchange chromatography. Discussion is facilitated by the use of less ambiguous terms, and for this reason a modified terminology proposed elsewhere ~ will be followed here. Adsorption dependent on biologically specific interactions (as illustrated diagrammatically in Fig. 1A) is termed bioallinity or biospecific adsorption. Adsorption which does not fall into this category is termed nonbiospecific adsorption rather than merely nonspecific adsorption. A similar distinction is applied in regard to the elution process, the term bioelution being applied to elution promoted by a soluble, biospecific 1Based in part on research supported by grants from the Medical Research Council and National Science Council of Ireland. 2p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968). 3p. O'Carra, in FEBS Symposium on "Industrial Aspects of Biochemistry" (B. Spenser, ed.), p. 107..North-Holland Publ., Amsterdam (1974).
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INTERFERENCE AND COMPLICATIONS
xI
~'n~-n~-n~-n~-n~-n~
Enzyme
109
A
"Def°rmingB'~buf~ CI [~
o>>
I o>
Fro. 1. Diagrammatic representations of (A) biospecific adsorption; (B) elution by a "deforming buffer"; (C) bioelution with a soluble, competitive counterligand. Im, immobilized ligand; C, competitive counterligand. counterligand competing with the immobilized one, as visualized in Fig. 1C. By contrast, nonbiospecific elution may involve gross physicochemical parameters (e.g., ionic strength-dependent elution from ion-exchange resins) or gross conformational changes in the enzyme caused by so-called "deforming buffers" as illustrated diagrammatically in Fig. lB. Although many workers have referred to the possibility of complicating nonbiospecific adsorption effects in affinity chromatographic systems, they have not always rigorously eliminated such interference. As a result, much of the affinity chromatography so far reported may be complicated by such phenomena. As shown below, it now appears that some important model studies probably depend largely, if not entirely, on nonbiospecific effects. The confusion of nonbiospecific with biospecific effects in the adsorption process is of more than academic interest and has considerable practical consequences. First, such confusion has resulted in the formulation of incorrect generalizations or rules-of-thumb which have to some extent adversely affected the design of new affinity chromatographic systems. 3,4 Apart from this, the failure to recognize or eliminate nonbiospecific complications early in the development of a particular affinity chromatographic system interferes directly with the main advantages that bioaffinity offers over conventional methods of purification. These anticipated advantages are: (a) rationality of design and operation, following a logic 4p. O'Carra, submitted to
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based on the known biological specificity of the enzyme to be purified; (b) ultraspecificity, deriving from the specificity of biological interactions. Interfering nonbiospecific adsorption may invalidate the rationale and affect the specificity in a largely unpredictable manner. Even though the operation of a system dominated by nonbiospecific adsorption may be adjustable to yield a reasonably good purification, the optimization of its efficiency will not be conducive to logic based on biospecificity. Even when the system is optimized by a trial-and-error approach, attempts to extend its use for example to the purification of the same enzyme from another source--will have no guarantee of success owing to the fact that the gross physicochemical properties of biologically homologous enzymes do not necessarily parallel their biospecificity. Such practical consequences are evident, for example, in the case of the system for ribonuclease A discussed below. It is even more crucial to distinguish rigorously between biospecific and nonbiospecific effects when an affinity chromatographic system is used as a tool for specificity studies or other enzyme-ligand binding studies in which the chromatographic behavior is interpreted in terms of active-site binding. 5,~ The care which must be exercised in this regard may be illustrated by some secondary applications of the affinity system developed for the purification of p-galactosidase. ~ As discussed under fl-galactosidase in the section on examples, the evidence now indicates that this system is largely, if not entirely, dependent on nonbiospecific interactions rather than on specific active-site binding. ~ This fact does not necessarily affect the value of this system in the original limited application for which it was developed (as a step in the purification of p-galactosidase from E s c h e r i c h i a c o l i ) , but it does seriously undermine any conclusions drawn from the chromatographic results on the assumption that these reflect active-sitespecific binding--as, for example, the interpretation of the chromatographic behavior of various mutant and fragmentary forms of p-galactosidase in terms of comparative active-site competence. .~ This p-galactosidase system has also played an important role in the development of the view that immobilized ligands with dissociation constants in the millimolar range are capable of promoting very strong specific adsorption, 1°,~1 and this view in turn has led to the development of the ~P. O'Carra and S. Barry, F E B S Lett. 21, 281 (1972). 6S. Barry and P. O'Carra, Biochern. J. 135, 595 (1973). rE. Steers, Jr., P. Cuatrecasas, and H. B. Pollard, J. Biol. Chem. 246, 196 (1971). 8p. O'Carra, S. Barry, and T. Griffin, Biochem. Soc. Trans. 1, 289-290 (1973). ° M. R. Villarejo and I. Zabin, Nattu'e (London) N e w Biol. 242, 50.52 (1973). 10p. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345. 11p. Cuatrecasas and C. B. Anfinsen, ,4nnu. Rev. Biochem. 40, 259 (1971).
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concept of "progressively perpetuating effectiveness. ''12 This concept is not consistent with much other evidence, or with the theoretical treatment summarized in the next section, and it is difficult to reconcile with kinetic theory in general. 3,' Again, the observations that largely gave rise to this concept are explainable in terms of nonbiospecific rather than biospecific adsorption. Detection of Nonbiospeeifie Interference A recently developed theoretical treatment of affinity chromatography has facilitated rigorous analysis. 3,4 The chief value of such analysis to date has been the uncovering of discrepancies that have been traced to the forms of interference discussed below. Some previous attempts at theoretical treatment failed to adequately reveal such discrepancies, either because the treatment TM consisted of the formulation of generalizations based on observations that were themselves complicated with nonbiospecific effects or because the treatment 13,l' failed to relate the enzyme-ligand binding in a useful or quantified manner with chromatographic parameters. Rigorous and useful analysis becomes possible only if the retardation of the enzyme by the column of adsorbent is quantified in units directly relevant to practical chromatographic operations, but free from dependence on such arbitrary and variable factors as the volume of the column and the rate of chromatographic irrigation. A simple and effective method of quantification is achieved by expressing the elution volume of the enzyme as a ratio relative to the operating volume of the column, after subtraction of the breakthrough volume. In other words, retardation ( R ) is made equivalent to the elution volume of the enzyme expressed in column-volume units, minus one unit representing the initial breakthrough volume through which even a completely unretarded substance must pass. When expressed thus, the retardation due solely to bioaffinity, Rblo, may be readily shown 4 to be related to the molar dissociation constant, K~.... and the effective molar concentration, [Im], of the immobilized ligand by the following equation. Rb~o = [Im]/Kim
(1)
Addition of a competitive counterligand to the irrigant will result in a decrease in the biospecific retardation depending on the relative concentrations and dissociation constants of the immobilized and soluble ligands. 12 p. Cuatrecasas, Advan. Enzymol. Relat. Areas Mol. Biol. 36, 29 (1972). I:'R. H. Reiner and A. Walch, Chromatographia 4, 578 (1971). a~ H. F. Hixson, Jr. and A. H. Nishikawa, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 1078 (Abstract) (1971).
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The resultant retardation, again assuming dependence only on pure bioaffinity, may be shown 4 to be described by the equation Kc where Kc and [C] are the dissociation constant and the concentration respectively of C, the soluble, competitive counterligand. Other theoretical considerations indicate that for multisubunit enzymes the immobilized state of the ligand should result effectively in a decrease in the dissociation constant, relative to that of an exactly equivalent soluble ligand, by a factor equal to the number of equivalent binding sites per enzyme molecule.' This appears to be the only complication introduced intrinsically by the immobilized state as such, and any other discrepancies between theoretical prediction and experimental observation are regarded as possible indications of interference. Negative discrepancies, i.e., retardations lower than predicted, are in general readily attributable either to occlusion of the ligand, usually by the matrix, resulting in a decrease in the accessibility (and hence in the effective concentration) of the ligand, or to steric hindrance by the immobilization linkage at the point of attachment of the ligand, resulting in an increase in the effective dissociation constant. Retardations which are considerably greater than predicted (after due allowance for any "multiple binding-site effect") have invariably been traced to interference from nonbiospecific adsorption effects. Such interference also results in anomalous bioelution behavior; in cases of severe interference, bioelution is usually not demonstrable at all (see below for examples). The principal reason for the initial lack of awareness of such interference in affinity chromatographic studies is probably attributable to the most commonly practiced methods of elution. These have most commonly employed so-called "deforming buffers," that is buffers of extreme pH, ionic strength, or other physicochemical property, which elute the macromolecule by deforming or distorting its conformation, thereby weakening or abolishing the chromatographic adsorption, supposedly as visualized in Fig. lB. Such methods of elution must be regarded as largely nonbiospecific in character, and they are much less logical (and less effective) than biospecific methods of elution using biospecific counterligands; a deforming buffer is just as likely to exert an effect on gross physicochemical interactions between enzyme and adsorbent as on biospecific ones. Indeed, in many cases, the eluting parameters bear no demonstrable relationship whatever to any known biospecific interactions of the enzyme being puri-
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fled, and the entire chromatographic process may easily depend on nonbiospecific effects rather than on bioaffinity. Thus in many studies the nonbiospecific nature of the elution process may hide a similar lack of biospecificity in the adsorption process. Examples of this effect are to be found in the first three examples in the section on examples, below. Sources and Nature of the Interference Until recently, ion-exchange effects seem to have been the only type of positive interference seriously considered by many workers (see, e.g., references 15 and 16), and it appears to have been widely believed that interference could be adequately controlled by eliminating ionic groups in the matrix material and in the spacer arms used. In many cases, however, the ligands themselves are ionic and may promote ion-exchange binding. Interference from this source seems possible in the affinity system for ribonuclease A, as outlined below in the section giving examples and in the chromatography of certain NAD-linked dehydrogenases described by Lowe and Dean ~7 as outlined in the examples section. However, studies with "mock affinity systems" indicate that the most common source of interference stems from the spacer arms in common use, in spite of their largely nonionic character. "Mock affinity systems" consist of analogs of the affinity gels with the biospecific ligand either absent or replaced by a close chemical analog having little or no biospecific affinity for the enzyme under consideration--see, for example, the investigation of the system for/3-galactosidase (section on examples, below) and for NAD-linked dehydrogenases (section on examples, below). Such studies have revealed widespread interference from the hydrocarbon spacer arms commonly in use. :~,s,l'~ The nature of the interfering adsorption has not as yet been fully elucidated, but the extent and magnitude of the interference correlates closely with the extent of the hydrophobic regions in the spacer arms. This suggests that the interference results largely from what are loosely termed hydrophobic interactions, and the elimination of much of the nonbiospecific binding by replacement of the hydrophobic spacer arms by polar or hydrophilic ones (as described below in the sections on control of interference and on examples) seems to confirm this interpretation. Hydrocarbon-substituted agarose gels have recently received attention 15p. D. G. Dean and C. R. Lowe, Biochem. J. 127, ll-12p (1972). ~6H. F. Hixson, Jr. and A. H. Nishikawa, Arch. Biochem. Biophys. 154, 501 (1973). 17C. R. Lowe and P. D. G. Dean, F E B S Lett. 14, 313 (1971). '" P. O'Carra, S. Barry, and T. Griffin, unpublished work (1973).
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as adsorbents in their own right for chromatography of proteins. 19-~2 Such "hydrophobic chromatography" has been regarded by some authors as a form of affinity chromatography, z1,~-~but the adsorption involved has been recognized in most cases as being basically nonbiospecific in character, and the development of such hydrophobic chromatography has independently drawn attention to the possible complications deriving from hydrocarbon spacer arms in bioaffinity chromatography. 23 In a number of cases, the nonbiospecific adsorption caused by predominantly hydrophobie spacer arms could be eliminated by raising the ionic strength '%'%6,~8 as well as by replacing the a r m by a polar one. The azo-NAD system for lactate dehydrogenase described below under examples typifies such behavior. It is generally considered that hydrophobic interactions should be strengthened rather than weakened by raising the ionic strength of the medium. Therefore the salt-sensitive nonbiospecific adsorption in such systems may involve other interactions, such as van der Waals and possibly electrostatic interactions of a nonionic nature. A further, and seemingly paradoxical, complication appears to be explainable only on the basis of nonbiospecific adsorption which does not become obtrusive unless the enzyme is brought into close contact with the spacer arm by prior biospecific adsorption on an attached ligand. The type of behavior which suggests such a complicated type of interference is exemplified by the case of glyceraldehyde-3-phosphate dehydrogenase discussed below. This phenomenon has been termed ligand-dependent nonbiospecific adsorption 3 and is characterized by the proportion of the enzyme which is bioelutable becoming progressively smaller the longer the enzyme is left adsorbed on the ligand-arm assembly. The enzyme seems to become progressively more enmeshed in the hydrophobic spacer arm the longer it is left in close proximity to it. '%" This effect also is abolished by replacing the arm with a hydrophilic counterpart as described below. One further type of interference may become operative where the spacer arm is long and flexible and capable of interacting noncovalently with the attached ligand. Molecular models have suggested in a number of such cases that the entire ligand-arm assembly might be capable of adopting a folded conformation in which the ligand "curls back" and interacts with the spacer arm, e.g., by hydrophobic interactions, in such a 'gR. J. Yon, Biochem. Y. 126, 765 (1972). ~°Z. Er-el, Y. Zaidenzaig, and S. Shaltiel, Biochem. Biophys. Res. Commun. 49, 383 (1972). See also this volume [9]. ~' B. H. J. Hofstee, Anal. Biochem. ,52, 430 (1973). 22p. Roschlau and B. Hess, Hoppe-Seyler's Z. Physiol. Chem. 353, 441 (1972). "~'~S. Shaltiel and Z. Er-el, Proc. Nat. Acad. Sci. U.S. 70, 778 (1973). See also this volume [9].
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way that it becomes masked or occluded. Such "conformational occlusion" has been suggested in explanation of the unexpected chromatographic ineffectiveness of certain affinity gels bearing hydrophobic ligands attached through hydrophobic spacer arms? .' Compound Affinity Although nonbiospecific adsorption is, in general, an undesirable complication in affinity chromatography, a number of cases have nov) been documented in which nonbiospecific interactions could be judiciously controlled in such a way as to usefully reinforce weak bioaffinities? ,~,x8 In such cases the operating conditions are carefully adjusted or balanced so as to allow a residual level of nonbiospecific interaction which is just sufficient to reinforce the weak bioaffinity synergistically without dominating or obscuring it. The reinforced bioaffinity behaves very much like strong bioaffinity, and the enzyme may be cleanly eluted with biospecific counterligands. This effect has been termed compound affinity? ,~ Examples are described below in the last section. Another example of such an effect is the reinforcement of the weak "abortive complex affinity" of the H-type isoenzyme of lactate dehydrogenase described elsewhere in the volume. 24 In such compound affinity it appears that the two types of weak interaction working in conjunction somehow cause strong adsorption of the enzyme, while either alone is incapable of causing more than weak retardation. Thus, when either is abolished, the enzyme is eluted. Such compound affinity is therefore readily distinguishable from pure bioaffinity by observing the effect on the chromatographic behavior produced by eliminating the nonbiospecific interactions completely. In the examples described in the last section, the reinforcing interactions seem to derive from the hydrophobic spacer arms as evidenced by the sensitivity of the effect to changes in the character, as distinct from the length, of these arms. It has been suggested 6 that in such systems a positive reinforcing effect may constitute a more important element in the role of the arm than the passive spacing effect. Although such reinforcement of weak bioaffinity can be very useful, the critical involvement of nonbiospecific interactions introduces some of the complications already discussed in the first section in relation to nonbiospecific adsorption effects generally. Since compound affinity involves a certain dependence on the gross physicochemical properties of the enzyme as well as on its bioaffinity, a method based on such compound
:4 p. O'Carra and S. Barry, this volume [77].
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affinity may not be automatically transferable to an homologous enzyme from another source. Control and Elimination of Interference Approaches to the control or elimination of nonbiospecific adsorption fall into two categories: (a) adjustment of operating conditions, (b) modification of the adsorbent. Only the former approach seems applicable where the interfering adsorption, as well as the bioaffinity, derives from the ligand itself, but in the majority of cases, where the interference derives from the spacer arms, both approaches are possible. The ionic strength of the irrigant has been found the most useful adjustable parameter in the operating conditions to date. 3,~,6 A salt, e.g., NaC1 or KC1, is simply added to the irrigant in increasing concentrations until satisfactory control of the nonbiospecific adsorption is achieved. Interfering ion-exchange adsorption is most obviously controllable by such means, but as mentioned in the preceding section, the interaction of some enzymes with spacer arms of apparently hydrophobic (and certainly nonionic) character may also be eliminated by increasing the ionic strength sufficiently. Examples of such control may be found in the next section and elsewhere in this volumeY ~ Where the bioaffinity of the system is weak, it may be more advantageous to allow the operation of compound affinity as discussed in the preceding section. This requires careful, and largely trial-and-error, balancing of the ionic strength rather than arbitrarily increasing it to a high level. Examples of such control, as distinct from elimination, of nonbiospecific interactions may also be found in the next section and elsewhere in this volume. 2~ While in certain cases the bioaffinity of enzymes for active-site ligands may be affected by changes in ionic strength, available kinetic evidence suggests that such cases are exceptional. Relatively high concentrations of NaC1 or KCI in particular have little effect on the specific ligand binding of many enzymes in spite of the seemingly widespread assumption to the contrary in the field of affinity chromatography--see, for example, the case of the adsorption of lactate dehydrogenase on ribosyl-linked NAD + discussed in the next section. By contrast, many other possible types of adjustment of the operating conditions--including, for example, pH and temperature changes--are more likely to disturb the bioaffinity and are less generally useful. Nevertheless, adjustment of the operating temperature has been found to be important in the perfection of at least one affinity chromatographic system. ~ In view of the temperature-dependence of at least some nonbiospecific
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CNBr - activated Sepharose 4 B 1, 3-diaminopropan-2-ol ~ '~
OH I
~--NH--CH2--CH-- CH2--NH2 bromoacetylation OH
O
--NH --C H 2 - C H - - C H 2 - N H -
C-- CH2Br
diaminopropanol o
~
--NH--CH2--CH--CH2--NH --C --CH2--NH-- CH~-- C H - - C H 2 - - N H 2
FIG. 2. Structures and method of preparation of typical polar or hydrophilic spacer arms. Beaded agarose gel (Sepharose 4B, Pharmacia) is activated with CNBr in the usual manner [P. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345; P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970)]. (See also this volume [21 and [6].) The washed, activated gel is quickly mixed with an equal volume of an ice-cold 3 M solution of 1,3-diaminopropan-2-ol adjusted to pH 10, and the mixture is stirred gently at 4 ° for about 20 hours. The resulting derivatized gel (I) is washed with about 40 volumes of 0.2 M saline followed by water, and it is then bromoacetylated as described by Cuatrecasas (loc. cit). The bromoacetylated gel is mixed with an equal volume of 0.4 M 1,3-diaminopropan-2-ol in 0.1 M NaHCO:, buffer adjusted to pH 9, and the mixture is stirred gently at room temperature (ca. 15 °) for 3 days. The gel is washed with 0.1 M saline (about 40 volumes) and with distilled water (about 10 volumes), and any unsubstituted bromoacetyl groups remaining are blocked by incubation of the gel with 0.2M mercaptoethanol in 0.l M NaHCO~ buffer, pH 9 at 15 ° for 24 hours followed by washing as before. The process of bromoacetylation followed by addition of diaminopropanol may be repeated to yield even longer arms. The gels may be further derivatized by a number of the general methods described throughout this volume so as to provide suitable termini for attachment of the ligands. Data from P. O'Carra, S. Barry, and T. Griffin, unpublished. a d s o r p t i o n effects and p r o b a b l y of m a n y bioaffinities, it should be b o r n e in mind that an affinity system which works well at one t e m p e r a t u r e m a y be m u c h less successful, or m a y require f u r t h er o p e r a t i o n a l adjustments, if the o p e r a t i n g t e m p e r a t u r e is c h a n g e d ? ,24 In several cases of a d s o r p t i o n of enzymes on h y d r o p h o b i c spacer arms, salt c o n c e n t r a t i o n s even a b o v e 1 M did n o t h i n g to a m e l i o r a t e the interf e r en ce (see e.g., the e x a m p l e s of /3-galactosidase and alcohol d e h y d r o genase in the next s e c t i o n ) . A t t e m p t s to c o u n t e r a c t such severe h y d r o -
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phobic interference by addition of organic solvents or urea to the irrigant have met with little success, mainly because adequate concentrations of these additives are also capable of denaturing the enzymes. However, the alternative general approach--modification of the adsorbent, particularly the spacer a r m s - - h a s shown promise with a number of such "sticky" enzymes. The most successful tactic has been to incorporate polar groups, e.g., carbinol, amido and secondary amino groups, at regular intervals along the length of the spacer arms so that no extensive hydrophobic region remains. Prototype examples of such hydrophilic arms are shown in Fig. 2. Evidence of the effectiveness of such modified spacer arms in eliminating nonbiospecific adsorption is to be found in examples devoted to fl-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase in the next section. Such hydrophilic spacer arms appear to eliminate nonbiospecific adsorption so completely that even the marginal level of such adsorption required as a reinforcing factor in compound affinity is also eliminated (cf., in next section, the examples in subsections on azo-linked NAD + and on the 6-1inked analogs of N A D + and A M P ) . Some Examples f l - G alactosidase
The affinity chromatographic system developed by Steers et al. 7 for fl-galactosidase employs a fl-thiogalactoside ligand attached to agarose via a spacer arm, as represented by structure (III). The method of elution is of the "deforming buffer" type and employs 0.1 M borate at pH 10. This affinity system promoted very strong adsorption of the enzyme even when the substitution level of the gel was as low as 0.6 mM, i.e., 0.6/zmole of ligand per milliliter of packed gel5 The long spacer arm of derivative (III) was found to be necessary for this strong adsorption, derivatives with shorter arms being ineffective. ~ This was interpreted purely in terms of elimination of steric hindrance, and these results have been widely quoted in support of the view that really long spacer arms may be necessary to eliminate steric hindrance. 7,l°-13,~-~ However, since the K~ value for the fl-thiogalactoside ligand is as high as 5 raM, r,'l there is an apparent wide discrepancy between the very weak retardation predicted by Eq. 1 (above) and the very strong adsorption observed with derivative ( I I I ) 2 , 9 Furthermore, satisfactory bioelution was not observed with this system2 ,7 "~P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
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AND COMPLICATIONS o
119
o
--NH--CH2-- CH2--CH~--NH--CHz--CH2--CH2--NH--C--CH2-- CH~--C --HN ~
S-~3-Galactoside
(III)
O O --NH--CH~--CH2 CHz--NH--CH~ CH2--CH2--NH--C--CH2--CH2--C--HN~---O-c~-GIucoside (iv)
~--NH--CH~
CH2--CH~--CH2--CH~--CH2--NH C--CH2--HN ~
(v) Nonbiospecific analogs of derivative (III) were thercfore prepared and studied. Derivative (IV) is an analog which differs from (III) in that the fl-galactoside ligand is rcplaced by an a-glucoside residue (for which fl-galactosidase should show little or no bioaffinity). Derivative (V) is a rcpresentative example of a number of "mock affinity gel" carrying no recognizable ligand at all. The chromatographic behavior of fl-galactosidase on dcdvativc (IV) was indistinguishable from its behavior on derivative (III) '~,ls and the enzyme was even more strongly adsorbed on the ligandless derivative (V) and on a numbcr of similar analogs. Elution could still be achieved with 0.1 M borate at pH 10, but only slowly and with much tailing2 ,'~ The enzyme was not eluted from any of the gels by biospecific countcrligands, i.e., soluble fl-galactosides, even when high salt concentrations were applied at the same time2 .s.'s The adsorption involved in this system therefore seems to be predominantly, attributable to nonbiospecific interactions involving the spacer arms. The hydrocarbon nature of these arms and the apparent ineffectiveness of high salt concentrations in counteracting the adsorption suggest that the interactions are of a hydrophobic nature. This seems to be confirmed by the fact that fl-galactosidase was not strongly adsorbed on gels carrying polar or hydrophilic spacer arms such as that of derivative (II) (Fig. 2). Ribonuclease A
The affinity gel developed by Wilchek and Gorecki 2G for ribonuclease A utilizes as adsorbent agarose-APUP, a uridine 2'(3'),5'-diphosphate ligand immobilized by attachment to agarose via an aminophenyl spacer group. The enzyme is satisfactorily adsorbed on this gel only when applied ~;M. Wilchek and M. Gorecki, Eur. J. Biochem. 11, 491 (1969). See also this volume [57].
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in dilute buffer at pH 5.2. In the original work elution was achieved with 0.2 M acetic acid, but elution can also be achieved by raising the pH to 7.5 at moderate ionic strength (0.1 to 0.2 M KCI) or by raising the ionic strength to a high level (0.5 to 1 M KC1) at pH 5.2. ~ The pH optimum of the enzyme is in the region of pH 7.0, and salt concentrations up to 0.2 M are not notably inhibitory. 2s It is therefore difficult to rationalize the adsorption and elution behavior satisfactorily in terms of pure bioaffinity. In view of the anionic nature of the ligand, due to its phosphate groups, the possibility that the adsorption may be complicated by nonbiospecific ion-exchange effects must be considered. This possibility is also suggested by the fact that the chromatographic behavior of bovine pancreatic ribonuclease A on simple anionic ion-exchange resins such as IRC-50 and CM-cellulose is in many respects similar to its behavior on the affinity gel; adsorption of the enzyme is strong at or near pH 5.2 and is weakened by raising the pH or by increasing the ionic strength. 2~,2'~ The enzyme may also be eluted--though much less satisfactorily--with aqueous acetic acid. z7 Difficulties have been reported by several workers who have used this affinity system in the purification of ribonucleases from sources other than beef pancreas. A ribonuclease from a myxomycete '~° and one of broad specificity from a shellfish source "~1 behaved quite differently from the bovine pancreatic enzyme; the purification achievable in these cases was not considered useful. Even pancreatic ribonuclease from other mammalian species behaved differently and modifications of the operating conditions had to be developed by a trial-and-error approach rather than by logic based on biospecificity. 3~ These difficulties are understandable if the adsorption and elution depend largely on gross electrostatic properties since these bear no necessary relationship to biospecificity. Adsorption of Lactate Dehydrogenase on Ribosyl-Linked N A D +
Lowe and Dean 17,'33 have described model affinity chromatographic studies of lactate dehydrogenase and other dehydrogenases on a "*~C. M a h o n and P. O'Carra, unpublished work. "°SF. M. Richards and H. W. Wyckoff, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. IV, p. 647. Academic Press, New York (1971). " C . H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953). '~°D. R. Farr, H. Amster, and M. Horisberger, Arch. Microbiol. 85, 249 (1972). 31W. Murphy and D. B. Johnson, Comp. Biochem. Physiol. in press; S. Bree, D. B. Johffson, and M. P. Coughlan, Comp. Biochem. Physiol. in press. 32R. K. Wierenga, J. D. Huizinga, W. Gaastra, G. W. Welling, and J. J. Beintema, F E B S Lett. 31, 181 (1973). 33C. R. Lowe and P. D. G. Dean, Biochem. J. 133, 515 (1973).
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number of immobilized N A D ~ preparations. One of these was prepared by direct coupling of N A D ~ to a CNBr-activated polysaccharide matrix and another by carbodiimide-promoted attachment of N A D + via the terminal carboxyl group of a caprylic acid-substituted polysaccharide matrix. It was initially assumed that the point,of attachment of the N A D ÷ in these derivatives was the 6-amino group of the adenine residue, 17 but it has since been established that attachment is mainly, if not entirely, through the ribosyl hydroxyl groups of the N A D + . 6,34 In the model studies of Lowe and Dean, 17,~'~ enzymes are applied in a buffer of low ionic strength and elution is achieved by a concentration gradient of KCI. These authors quantify affinity chromatographic effectiveness in terms of "binding" which is defined as the millimolar salt concentration needed to elute an enzyme. On the basis of this criterion both of the above-mentioned immobilized N A D ÷ derivatives were considered to be effective affinity systems for lactate dehydrogenase. However, kinetic studies show that the KC1 concentrations used for elution of this enzyme have little or no effect on the biospecific binding of soluble N A D ÷ to lactate dehydrogenase. ~ Moreover, at the low ionic strength of the starting buffer the adsorbed enzyme is not susceptible to bioelution by soluble N A D ~ added to the irrigant. This evidence suggests that the adsorption-elution behavior observed with these derivatives is dominated by salt-sensitive, nonbiospecific adsorption. Dean and Lowe 15 and Lowe et al. ~ have suggested that the CNBr activation step introduces ionic groups into the matrix which must be neutralized or eliminated before satisfactory results are obtainable. However the N A D + residue itself is ionic and would be expected to promote some ion-exchange adsorption at low ionic strength. This presumably dominates the chromatography, since at most only very weak bioaffinity can be detected with the ribosyl-linked N A D ÷ derivatives under discussion2 This is consistent with the detailed picture of the NAD~-binding site of lactate dehydrogenase which has recently emerged from the X-ray diffraction studies of Rossman and co-workers'~,~: both ribosyl residues of the N A D + molecule are buried in the binding crevice and are involved in the binding interactions of N A D ~ with the enzyme. Attachment of the N A D ÷ molecule ~' K. Mosbach, H. Guilford, R. Ohlsson, and M. Scott, Biochem. J. 127, 625 (1972). :~C. R. Lowe, M. J. Harvey, D. B. Craven, and P. D. G. Dean, Biochem. J. 133, 499 (1973). ~" M. G. Rossman, M. J. Adams, M. Buehner, G. C. Ford, M. L. Hackert, P. 1. Lentz, Jr., A. McPherson, Jr., R. W. Schevitz, and I. E. Smiley, Cold Spring Harbor Symp. Quant. Biol. 36, 179 (1972). '~M. J. Adams, M. Buchner, K. Chandrasekhar, G. C. Ford, M. L. Hackert, A. Liljas, M. G. Rossman, I. E. Smiley, W. S. Allison, J. Everse, N. O. Kaplan, and S. S. Taylor, Proc. Nat. Acad. Sci. U.S. 70, 1968 (1973).
122
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REACTIONS
AND
GENERAL
METHODOLOGY
[8]
through either of these residues would therefore be expected to drastically decrease or abolish the bioaffinity of the resulting derivative. Azo-Linked
NAD +
Immobilized N A D + derivatives may also be prepared by incubation of N A D + with diazotized derivatives of matrix-spacer arm assemblies such as derivative ( V I ) . 6 The N A D + is thought to attach through the 8-position of the adenine residue. 6'~s"Immobilized N A D P + analogs can be similarly prepared, 6 and N A D + has been similarly attached via the hydrophilic spacer arms described above in the section on control of interference. 18 A group of such derivatives, ( V I ) to ( I X ) , have been used for affinity chromatographic studies of a range of NAD+-linked dehydrogenases, and these studies have provided useful insights into some of the complications that can be encountered in affinity chromatography. 6,1s // - - NH-- CH 2 - CH z - CH2- - CH z - - CH 2- - CH 2 - NH -- C ~ x
\\ ~-NH2
(vi)
--NH --CH2--CH2--CH2--CH2--CH2--CH2--NH --C -~<
~
N----N--NAD
(vii)
~--NH--CH2--CH2--CH2--CH~--CH2--CH2--NH--~~N~N--NADP + (vm)
OH
0 II
OH l
0 )I
#
\\
--NH--CH2--CH--CH2--NH--C--CH2--NH--CH2--CH--CH2--NH--C ~ - - N ~ N
NAD +
(LX)
Yeast ,41coholDehydrogenasefi,IsThis
enzyme was firmly adsorbed on
derivative ( V I I ) , but it could neither be eluted by addition of soluble N A D + to the irrigant nor by KC1 concentrations as high as 1 M. Addition of both N A D + and high salt concentrations to the irrigant simultaneously also failed to elute the enzyme. On the other hand, the enzyme showed no detectable affinity for the hydrophilic derivative ( I X ) . It thus seems likely that the adsorption on ( V I I ) is predominantly or entirely due to interac3s M . K . W e i b e l , H . H . W e e t a l l , a n d H . ] . B r i g h t , 44, 347 ( 1 9 7 1 ) .
Biochem. Biophys. Res. Commun.
[8]
INTERFERENCE AND COMPLICATIONS
123
tions with the hydrophobic spacer arm of this derivative. The enzyme is also adsorbed on a number of agarose derivatives carrying hydrophobic groups and no biospecific ligand. TM Glyceraldehyde-3-phosphate Dehydrogenase. '~,1~ The enzyme from rabbit muscle is adsorbed strongly on derivative (VII). The adsorption is not noticeably affected by high concentrations of KC1, and the enzyme is cleanly eluted by 2 mM NAD + if a salt concentration of at least 0.2 M is maintained in the irrigant. Under such conditions the enzyme is not adsorbed on either the ligandless analog ( V I ) or on the NADP + analog (VIII). These results indicate clearly the biospeeific nature of the adsorption of this enzyme on derivative (VII). However, the yield of the enzyme elutable with NAD ~ becomes progressively lower the longer the enzyme is left adsorbed on the gel before elution. This loss is far in excess of the loss of activity in control experiments when samples of the enzyme are put through equivalent procedures on a "blank gel" such as derivative (VI). The hypothesis that this time-dependent loss of activity is due to slow nonbiospecifie adsorption onto the hydrophobic spacer arm, after the enzyme has first bound biospecifically to the immobilized NAD + is supported by the results obtained when the spacer arm is replaced by a hydrophilic one as in derivative ( I X ) . The enzyme is just as strongly adsorbed on this derivative as on derivative ( V I I ) , but when it is eluted with NAD ÷, the yield is greater than 90% and does not decrease significantly even when the enzyme is left on the column for 1.5 hours, by which time the yield of NAD+-elutable enzyme from derivative (VII) has decreased to about 10%. Lactate Dehydrogenase. 6,1s When lactate dehydrogenase is chromatographed at low ionic strength, e.g., in 0.05 M potassium phosphate at pH 7.4, it is strongly adsorbed on derivative ( V I I ) , but is not bioelutable; it is similarly adsorbed onto the ligandless analog (VI). At low ionic strength, therefore, the adsorption is dominated by nonbiospecific adsorption attributable to the matrix-spacer arm assembly. Inclusion of 0.5 M KC1 abolishes such interfering adsorption, as evidenced by the lack of adsorption of the enzyme on derivatives (VI) and (VIII) under such conditions. However, at such a high salt concentration the enzyme remains only weakly retarded by derivative (VII). Since such salt concentrations do not interfere significantly with the binding of soluble NAD ÷ to lactate dehydrogenase, 6 this result suggests that, although the azo-linked NAD + derivative retains some bioaffinity for this enzyme, it is rather weak, presumably as a result of partial steric hindrance by the immobilization linkage. This seems to be confirmed by the fact that lactate dehydrogenase
124-
COUPLING
REACTIONS
AND
GENERAL
METHODOLOGY
[8]
o --NH --CH~
C H 2 - - C H 2 - - C H 2 - - CH2-- C H 2 - - N H - - C - - C H z S
N
O
N
C--NH O-
O-
l /O--CH,--O--Ip--o--I--o--CH, O. I HO
OH
HO
OH
(x)
i
o - - N H -- C H 2 - C H 2-- CIl2-- C H 2-- Cl£z-- C H 2- - N H -- C -- C H 2
N
N
O-
HO
OH
(xl) OH I
0
OH
I¢
--NH--CH2--CH--CH2--NH--C--
I
CH2--NH--CHz--CH--CH~--NH--CO--
CH 2 S
OCH2--O--~?O
HO
OH
(Xm
shows only very weak affinity for derivative ( I X ) at salt concentrations as low as 0.1 M. The inclusion of 0.2 M KC1 in the irrigating buffer was sufficient to largely eliminate the nonbiospecific adsorption of this enzyme on derivatives (VI) and (VIII). At this intermediate ionic strength, lactate dehydrogenase was strongly adsorbed on derivative (VII), but not on derivative ( I X ) , and could be cleanly eluted with the biospecific counterligands N A D H and 5'-AMP (a weak competitive inhibitor against NAD+). This seemingly clear-cut bioaffinity is of a much stronger nature than could be accounted for by bioaffinity alone, in view of the results at high ionic strength and those with the hydrophilic spacer arm. The discrepancy is most plausibly explained on the basis of the compound affinity concept discussed in a previous section.
[8]
INTERFERENCE AND COMPLICATIONS
125
Analogs o[ N A D ÷ and A M P Linked through the 6 Position o[ the Purine Residue ls,39
Derivatives (X) to ( X I I ) may be regarded as immobilized thio analogs of NAD + and 5'-AMP linked through the 6-position of the purine residue. They are easily prepared by reacting the 6-mercaptopurine analogs of the nucleotides with bromoacetyl-terminating matrix-arm assemblies. '~9 This linkage procedure was considered promising for affinity chromatography of certain NAD-linked dehydrogenases, including lactate dehydrogenase, because it was known from kinetic studies that derivatization at the 6-position does not drastically affect binding of NAD + to these enzymes. Mosbach et al. 34,4°,41 have synthesized 6-1inked derivatives by another method on the basis of the same rationale. Lactate de hydrogenase was strongly adsorbed on the 6-immobilized NAD + analog ( X ) ? 9 Bioelution with soluble N A D + was clear-cut if the salt concentration in the irrigant was maintained above 0.1 M and the affinity remained strong at high ionic strengths (e.g., 0.5 M KCI) indicating that the strong affinity of derivative (X) for this enzyme is probably pure bioaffinity. This contrasts with the behavior of the azo-linked derivative (VII) which binds lactate dehydrogenase strongly only at moderate ionic strengths, apparently with the help of nonbiospecific reinforcement (see above). 5'-AMP is a weak competitive inhibitor of lactate dehydrogenase (K~ = ca. 2 mM). At the substitution level of the batches of derivative (XI) prepared to date ( < 1 mM), only very weak biospecific retardation would be predicted from Eq. 1. In agreement with this, the enzyme was only marginally adsorbed on derivative ( X I ) at high ionic strength (0.5 M KC1). However, at low ionic strength the enzyme was strongly adsorbed on this derivative and the ionic strength could be adjusted to intermediate values (e.g., 0.1 M) such that the enzyme remained strongly adsorbed but could be cleanly eluted with biospecific counterligands, such as 5'AMP and NADH. On the hydrophilic derivative (XII) the enzyme remained only marginally adsorbed at such intermediate ionic strength demonstrating the positive contribution of the hydrophobic arm of derivative ( X I ) to the compound affinity. Malate dehydrogenase is the least "sticky" enzyme so far encountered in our work. The enzyme from beef heart is only marginally retarded by hydrophobic "mock affinity gels," such as derivative (VI), even at low ionic strengths. This enzyme displays no detectable affinity for the azo:'"S. Barry and P. O'Carra, FEBS Lett. 37, 134 (1973). '°R. Ohlsson, P. Brodelius, and K. Mosbach, FEBS Lett. 25, 234 (1972). ~1K. Mosbach, this volume [16].
126
COUPLINGREACTIONS AND GENERAL METHODOLOGY
[9]
linked N A D + derivative ( V I I ) but is strongly adsorbed on the 6-1inked N A D ÷ analog ( X ) at moderate ionic strengths (0.1-0.2 M ) and is cleanly bioelutable with N A D H . Malate dehydrogenase shows only very weak affinity for the 6-1inked A M P analog ( X I ) and, in contrast to lactate dehydrogenase, this affinity is not reinforced at moderate ionic strengths, presumably owing to the general lack of nonbiospecific interaction of this enzyme with hydrophobic spacer arms. 4~ ,5 p. O'Carra and S. Barry, unpublished work (1973).
[9] Hydrophobic
Chromatography
B y SHMUEL SHALTIEL
D e v e l o p m e n t of the Basic Principle In the course of our studies on enzymes involved in glycogen metabolism, we attempted to coat beaded agarose with glycogen and use it for affinity chromatography 1 of these enzymes. The procedure used for the preparation of the columns consisted of: (a) activation of the agarose with CNBr z and reaction with an a,,0-diaminoalkane to obtain an ,o-aminoalkyl agarose, ( b ) binding of CNBr-activated glycogen to the o,-aminoalkyl agarose. Two glycogen-coated agarose preparations which differed only in the length of the hydrocarbon chains bridging the ligand and the bead (Seph-C8-NH-glycogen 3 and Seph-C4-NH-glycogen) displayed unexpected differences in the retention of glycogen phosphorylase b. Whereas the column with 8-carbon-atom bridges adsorbed the enzyme, the column with 4-carbon-atom bridges did not even retard it. 4 Considering the large molecular dimensions of glycogen (Fig. 1 ), it did not seem likely that changing the length of the extensions from 8 to 4 carbon atoms would have such a dramatic effect on the retention power of the column. Rather, we were initially inclined to think that the column with the shorter side chains had failed to bind glycogen because of a reduced accessibility of the amino groups. This possibility was excluded by repeating the syn1p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968). ~R. Ax6n, J. Porath, and S. Ernb~ick, Nature (London) 214, 1302 (1967). ~Seph-C~-NH-glycogen represents Sepharose 4B activated with CNBr, reacted With an ~,~-diaminoalkane n-carbon atoms long, then coupled with CNBr-activated glycogen. 4Z. Er-el, Y. Zaidenzaig, and S. Shaltiel, Biochem. Biophys. Res. Commun. 49, 383 (1972).
COUPLINGREACTIONS AND GENERAL METHODOLOGY
[8]
nium agarose, stirred in an ice bath. Formation of the colored azo gel begins immediately, and coupling is usually complete in 1 0 - 3 0 minutes. During this time, the mixture is allowed to warm to room temperature. If desired, any unreacted diazonium groups '~° may be blocked at this point by addition of a dilute solution of fl-naphthol or of 2,6-di-tert-butylphenol in 5 0 % dimethylformamide. The gel is filtered and washed thoroughly with water, with 50% dimethylformamide, and again with water, and is stored (cold) as a suspension in water or buffer. ~*This procedure is usually unnecessary for small ligands, especially when used in moderate excess in the coupling reaction.
[8] Interfering and Complicating
Adsorption
in Bioaffinity Chromatography
Effects
1
B y P.~DRAIG O'CARRA, STANDISH BARRY, and TADHG GRIFFIN
The term affinity chromatography was originally introduced to denote adsorption chromatography based on enzymatically specific interactions, -~ as distinct from adsorption dependent on gross physicochemical properties, which in this context has generally been referred to as nonspecific adsorption. This terminology is not entirely satisfactory since the term affinity could be validly applied to any form of adsorption, specific or otherwise, while the so-called nonspecific adsorption effects often have quite a high degree of apparent specificity, as evidenced by the enzyme purifications achievable by ion-exchange chromatography. Discussion is facilitated by the use of less ambiguous terms, and for this reason a modified terminology proposed elsewhere ~ will be followed here. Adsorption dependent on biologically specific interactions (as illustrated diagrammatically in Fig. 1A) is termed bioallinity or biospecific adsorption. Adsorption which does not fall into this category is termed nonbiospecific adsorption rather than merely nonspecific adsorption. A similar distinction is applied in regard to the elution process, the term bioelution being applied to elution promoted by a soluble, biospecific 1Based in part on research supported by grants from the Medical Research Council and National Science Council of Ireland. 2p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968). 3p. O'Carra, in FEBS Symposium on "Industrial Aspects of Biochemistry" (B. Spenser, ed.), p. 107..North-Holland Publ., Amsterdam (1974).
[8]
INTERFERENCE AND COMPLICATIONS
xI
~'n~-n~-n~-n~-n~-n~
Enzyme
109
A
"Def°rmingB'~buf~ CI [~
o>>
I o>
Fro. 1. Diagrammatic representations of (A) biospecific adsorption; (B) elution by a "deforming buffer"; (C) bioelution with a soluble, competitive counterligand. Im, immobilized ligand; C, competitive counterligand. counterligand competing with the immobilized one, as visualized in Fig. 1C. By contrast, nonbiospecific elution may involve gross physicochemical parameters (e.g., ionic strength-dependent elution from ion-exchange resins) or gross conformational changes in the enzyme caused by so-called "deforming buffers" as illustrated diagrammatically in Fig. lB. Although many workers have referred to the possibility of complicating nonbiospecific adsorption effects in affinity chromatographic systems, they have not always rigorously eliminated such interference. As a result, much of the affinity chromatography so far reported may be complicated by such phenomena. As shown below, it now appears that some important model studies probably depend largely, if not entirely, on nonbiospecific effects. The confusion of nonbiospecific with biospecific effects in the adsorption process is of more than academic interest and has considerable practical consequences. First, such confusion has resulted in the formulation of incorrect generalizations or rules-of-thumb which have to some extent adversely affected the design of new affinity chromatographic systems. 3,4 Apart from this, the failure to recognize or eliminate nonbiospecific complications early in the development of a particular affinity chromatographic system interferes directly with the main advantages that bioaffinity offers over conventional methods of purification. These anticipated advantages are: (a) rationality of design and operation, following a logic 4p. O'Carra, submitted to
Biochem. J.
110
COUPLING REACTIONS AND GENERAL METHODOLOGY
[8]
based on the known biological specificity of the enzyme to be purified; (b) ultraspecificity, deriving from the specificity of biological interactions. Interfering nonbiospecific adsorption may invalidate the rationale and affect the specificity in a largely unpredictable manner. Even though the operation of a system dominated by nonbiospecific adsorption may be adjustable to yield a reasonably good purification, the optimization of its efficiency will not be conducive to logic based on biospecificity. Even when the system is optimized by a trial-and-error approach, attempts to extend its use for example to the purification of the same enzyme from another source--will have no guarantee of success owing to the fact that the gross physicochemical properties of biologically homologous enzymes do not necessarily parallel their biospecificity. Such practical consequences are evident, for example, in the case of the system for ribonuclease A discussed below. It is even more crucial to distinguish rigorously between biospecific and nonbiospecific effects when an affinity chromatographic system is used as a tool for specificity studies or other enzyme-ligand binding studies in which the chromatographic behavior is interpreted in terms of active-site binding. 5,~ The care which must be exercised in this regard may be illustrated by some secondary applications of the affinity system developed for the purification of p-galactosidase. ~ As discussed under fl-galactosidase in the section on examples, the evidence now indicates that this system is largely, if not entirely, dependent on nonbiospecific interactions rather than on specific active-site binding. ~ This fact does not necessarily affect the value of this system in the original limited application for which it was developed (as a step in the purification of p-galactosidase from E s c h e r i c h i a c o l i ) , but it does seriously undermine any conclusions drawn from the chromatographic results on the assumption that these reflect active-sitespecific binding--as, for example, the interpretation of the chromatographic behavior of various mutant and fragmentary forms of p-galactosidase in terms of comparative active-site competence. .~ This p-galactosidase system has also played an important role in the development of the view that immobilized ligands with dissociation constants in the millimolar range are capable of promoting very strong specific adsorption, 1°,~1 and this view in turn has led to the development of the ~P. O'Carra and S. Barry, F E B S Lett. 21, 281 (1972). 6S. Barry and P. O'Carra, Biochern. J. 135, 595 (1973). rE. Steers, Jr., P. Cuatrecasas, and H. B. Pollard, J. Biol. Chem. 246, 196 (1971). 8p. O'Carra, S. Barry, and T. Griffin, Biochem. Soc. Trans. 1, 289-290 (1973). ° M. R. Villarejo and I. Zabin, Nattu'e (London) N e w Biol. 242, 50.52 (1973). 10p. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345. 11p. Cuatrecasas and C. B. Anfinsen, ,4nnu. Rev. Biochem. 40, 259 (1971).
[8]
INTERFERENCE AND COMPLICATIONS
111
concept of "progressively perpetuating effectiveness. ''12 This concept is not consistent with much other evidence, or with the theoretical treatment summarized in the next section, and it is difficult to reconcile with kinetic theory in general. 3,' Again, the observations that largely gave rise to this concept are explainable in terms of nonbiospecific rather than biospecific adsorption. Detection of Nonbiospeeifie Interference A recently developed theoretical treatment of affinity chromatography has facilitated rigorous analysis. 3,4 The chief value of such analysis to date has been the uncovering of discrepancies that have been traced to the forms of interference discussed below. Some previous attempts at theoretical treatment failed to adequately reveal such discrepancies, either because the treatment TM consisted of the formulation of generalizations based on observations that were themselves complicated with nonbiospecific effects or because the treatment 13,l' failed to relate the enzyme-ligand binding in a useful or quantified manner with chromatographic parameters. Rigorous and useful analysis becomes possible only if the retardation of the enzyme by the column of adsorbent is quantified in units directly relevant to practical chromatographic operations, but free from dependence on such arbitrary and variable factors as the volume of the column and the rate of chromatographic irrigation. A simple and effective method of quantification is achieved by expressing the elution volume of the enzyme as a ratio relative to the operating volume of the column, after subtraction of the breakthrough volume. In other words, retardation ( R ) is made equivalent to the elution volume of the enzyme expressed in column-volume units, minus one unit representing the initial breakthrough volume through which even a completely unretarded substance must pass. When expressed thus, the retardation due solely to bioaffinity, Rblo, may be readily shown 4 to be related to the molar dissociation constant, K~.... and the effective molar concentration, [Im], of the immobilized ligand by the following equation. Rb~o = [Im]/Kim
(1)
Addition of a competitive counterligand to the irrigant will result in a decrease in the biospecific retardation depending on the relative concentrations and dissociation constants of the immobilized and soluble ligands. 12 p. Cuatrecasas, Advan. Enzymol. Relat. Areas Mol. Biol. 36, 29 (1972). I:'R. H. Reiner and A. Walch, Chromatographia 4, 578 (1971). a~ H. F. Hixson, Jr. and A. H. Nishikawa, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 30, 1078 (Abstract) (1971).
112
COUPLING REACTIONS AND GENERAL METHODOLOGY
[8]
The resultant retardation, again assuming dependence only on pure bioaffinity, may be shown 4 to be described by the equation Kc where Kc and [C] are the dissociation constant and the concentration respectively of C, the soluble, competitive counterligand. Other theoretical considerations indicate that for multisubunit enzymes the immobilized state of the ligand should result effectively in a decrease in the dissociation constant, relative to that of an exactly equivalent soluble ligand, by a factor equal to the number of equivalent binding sites per enzyme molecule.' This appears to be the only complication introduced intrinsically by the immobilized state as such, and any other discrepancies between theoretical prediction and experimental observation are regarded as possible indications of interference. Negative discrepancies, i.e., retardations lower than predicted, are in general readily attributable either to occlusion of the ligand, usually by the matrix, resulting in a decrease in the accessibility (and hence in the effective concentration) of the ligand, or to steric hindrance by the immobilization linkage at the point of attachment of the ligand, resulting in an increase in the effective dissociation constant. Retardations which are considerably greater than predicted (after due allowance for any "multiple binding-site effect") have invariably been traced to interference from nonbiospecific adsorption effects. Such interference also results in anomalous bioelution behavior; in cases of severe interference, bioelution is usually not demonstrable at all (see below for examples). The principal reason for the initial lack of awareness of such interference in affinity chromatographic studies is probably attributable to the most commonly practiced methods of elution. These have most commonly employed so-called "deforming buffers," that is buffers of extreme pH, ionic strength, or other physicochemical property, which elute the macromolecule by deforming or distorting its conformation, thereby weakening or abolishing the chromatographic adsorption, supposedly as visualized in Fig. lB. Such methods of elution must be regarded as largely nonbiospecific in character, and they are much less logical (and less effective) than biospecific methods of elution using biospecific counterligands; a deforming buffer is just as likely to exert an effect on gross physicochemical interactions between enzyme and adsorbent as on biospecific ones. Indeed, in many cases, the eluting parameters bear no demonstrable relationship whatever to any known biospecific interactions of the enzyme being puri-
[8]
INTERFERENCE AND COMPLICATIONS
113
fled, and the entire chromatographic process may easily depend on nonbiospecific effects rather than on bioaffinity. Thus in many studies the nonbiospecific nature of the elution process may hide a similar lack of biospecificity in the adsorption process. Examples of this effect are to be found in the first three examples in the section on examples, below. Sources and Nature of the Interference Until recently, ion-exchange effects seem to have been the only type of positive interference seriously considered by many workers (see, e.g., references 15 and 16), and it appears to have been widely believed that interference could be adequately controlled by eliminating ionic groups in the matrix material and in the spacer arms used. In many cases, however, the ligands themselves are ionic and may promote ion-exchange binding. Interference from this source seems possible in the affinity system for ribonuclease A, as outlined below in the section giving examples and in the chromatography of certain NAD-linked dehydrogenases described by Lowe and Dean ~7 as outlined in the examples section. However, studies with "mock affinity systems" indicate that the most common source of interference stems from the spacer arms in common use, in spite of their largely nonionic character. "Mock affinity systems" consist of analogs of the affinity gels with the biospecific ligand either absent or replaced by a close chemical analog having little or no biospecific affinity for the enzyme under consideration--see, for example, the investigation of the system for/3-galactosidase (section on examples, below) and for NAD-linked dehydrogenases (section on examples, below). Such studies have revealed widespread interference from the hydrocarbon spacer arms commonly in use. :~,s,l'~ The nature of the interfering adsorption has not as yet been fully elucidated, but the extent and magnitude of the interference correlates closely with the extent of the hydrophobic regions in the spacer arms. This suggests that the interference results largely from what are loosely termed hydrophobic interactions, and the elimination of much of the nonbiospecific binding by replacement of the hydrophobic spacer arms by polar or hydrophilic ones (as described below in the sections on control of interference and on examples) seems to confirm this interpretation. Hydrocarbon-substituted agarose gels have recently received attention 15p. D. G. Dean and C. R. Lowe, Biochem. J. 127, ll-12p (1972). ~6H. F. Hixson, Jr. and A. H. Nishikawa, Arch. Biochem. Biophys. 154, 501 (1973). 17C. R. Lowe and P. D. G. Dean, F E B S Lett. 14, 313 (1971). '" P. O'Carra, S. Barry, and T. Griffin, unpublished work (1973).
114
COUPLINGREACTIONS AND GENERAL METHODOLOGY
[8]
as adsorbents in their own right for chromatography of proteins. 19-~2 Such "hydrophobic chromatography" has been regarded by some authors as a form of affinity chromatography, z1,~-~but the adsorption involved has been recognized in most cases as being basically nonbiospecific in character, and the development of such hydrophobic chromatography has independently drawn attention to the possible complications deriving from hydrocarbon spacer arms in bioaffinity chromatography. 23 In a number of cases, the nonbiospecific adsorption caused by predominantly hydrophobie spacer arms could be eliminated by raising the ionic strength '%'%6,~8 as well as by replacing the a r m by a polar one. The azo-NAD system for lactate dehydrogenase described below under examples typifies such behavior. It is generally considered that hydrophobic interactions should be strengthened rather than weakened by raising the ionic strength of the medium. Therefore the salt-sensitive nonbiospecific adsorption in such systems may involve other interactions, such as van der Waals and possibly electrostatic interactions of a nonionic nature. A further, and seemingly paradoxical, complication appears to be explainable only on the basis of nonbiospecific adsorption which does not become obtrusive unless the enzyme is brought into close contact with the spacer arm by prior biospecific adsorption on an attached ligand. The type of behavior which suggests such a complicated type of interference is exemplified by the case of glyceraldehyde-3-phosphate dehydrogenase discussed below. This phenomenon has been termed ligand-dependent nonbiospecific adsorption 3 and is characterized by the proportion of the enzyme which is bioelutable becoming progressively smaller the longer the enzyme is left adsorbed on the ligand-arm assembly. The enzyme seems to become progressively more enmeshed in the hydrophobic spacer arm the longer it is left in close proximity to it. '%" This effect also is abolished by replacing the arm with a hydrophilic counterpart as described below. One further type of interference may become operative where the spacer arm is long and flexible and capable of interacting noncovalently with the attached ligand. Molecular models have suggested in a number of such cases that the entire ligand-arm assembly might be capable of adopting a folded conformation in which the ligand "curls back" and interacts with the spacer arm, e.g., by hydrophobic interactions, in such a 'gR. J. Yon, Biochem. Y. 126, 765 (1972). ~°Z. Er-el, Y. Zaidenzaig, and S. Shaltiel, Biochem. Biophys. Res. Commun. 49, 383 (1972). See also this volume [9]. ~' B. H. J. Hofstee, Anal. Biochem. ,52, 430 (1973). 22p. Roschlau and B. Hess, Hoppe-Seyler's Z. Physiol. Chem. 353, 441 (1972). "~'~S. Shaltiel and Z. Er-el, Proc. Nat. Acad. Sci. U.S. 70, 778 (1973). See also this volume [9].
[8]
INTERFERENCE AND COMPLICATIONS
115
way that it becomes masked or occluded. Such "conformational occlusion" has been suggested in explanation of the unexpected chromatographic ineffectiveness of certain affinity gels bearing hydrophobic ligands attached through hydrophobic spacer arms? .' Compound Affinity Although nonbiospecific adsorption is, in general, an undesirable complication in affinity chromatography, a number of cases have nov) been documented in which nonbiospecific interactions could be judiciously controlled in such a way as to usefully reinforce weak bioaffinities? ,~,x8 In such cases the operating conditions are carefully adjusted or balanced so as to allow a residual level of nonbiospecific interaction which is just sufficient to reinforce the weak bioaffinity synergistically without dominating or obscuring it. The reinforced bioaffinity behaves very much like strong bioaffinity, and the enzyme may be cleanly eluted with biospecific counterligands. This effect has been termed compound affinity? ,~ Examples are described below in the last section. Another example of such an effect is the reinforcement of the weak "abortive complex affinity" of the H-type isoenzyme of lactate dehydrogenase described elsewhere in the volume. 24 In such compound affinity it appears that the two types of weak interaction working in conjunction somehow cause strong adsorption of the enzyme, while either alone is incapable of causing more than weak retardation. Thus, when either is abolished, the enzyme is eluted. Such compound affinity is therefore readily distinguishable from pure bioaffinity by observing the effect on the chromatographic behavior produced by eliminating the nonbiospecific interactions completely. In the examples described in the last section, the reinforcing interactions seem to derive from the hydrophobic spacer arms as evidenced by the sensitivity of the effect to changes in the character, as distinct from the length, of these arms. It has been suggested 6 that in such systems a positive reinforcing effect may constitute a more important element in the role of the arm than the passive spacing effect. Although such reinforcement of weak bioaffinity can be very useful, the critical involvement of nonbiospecific interactions introduces some of the complications already discussed in the first section in relation to nonbiospecific adsorption effects generally. Since compound affinity involves a certain dependence on the gross physicochemical properties of the enzyme as well as on its bioaffinity, a method based on such compound
:4 p. O'Carra and S. Barry, this volume [77].
116
COUPLING REACTIONS AND GENERAL METHODOLOGY
[8]
affinity may not be automatically transferable to an homologous enzyme from another source. Control and Elimination of Interference Approaches to the control or elimination of nonbiospecific adsorption fall into two categories: (a) adjustment of operating conditions, (b) modification of the adsorbent. Only the former approach seems applicable where the interfering adsorption, as well as the bioaffinity, derives from the ligand itself, but in the majority of cases, where the interference derives from the spacer arms, both approaches are possible. The ionic strength of the irrigant has been found the most useful adjustable parameter in the operating conditions to date. 3,~,6 A salt, e.g., NaC1 or KC1, is simply added to the irrigant in increasing concentrations until satisfactory control of the nonbiospecific adsorption is achieved. Interfering ion-exchange adsorption is most obviously controllable by such means, but as mentioned in the preceding section, the interaction of some enzymes with spacer arms of apparently hydrophobic (and certainly nonionic) character may also be eliminated by increasing the ionic strength sufficiently. Examples of such control may be found in the next section and elsewhere in this volumeY ~ Where the bioaffinity of the system is weak, it may be more advantageous to allow the operation of compound affinity as discussed in the preceding section. This requires careful, and largely trial-and-error, balancing of the ionic strength rather than arbitrarily increasing it to a high level. Examples of such control, as distinct from elimination, of nonbiospecific interactions may also be found in the next section and elsewhere in this volume. 2~ While in certain cases the bioaffinity of enzymes for active-site ligands may be affected by changes in ionic strength, available kinetic evidence suggests that such cases are exceptional. Relatively high concentrations of NaC1 or KCI in particular have little effect on the specific ligand binding of many enzymes in spite of the seemingly widespread assumption to the contrary in the field of affinity chromatography--see, for example, the case of the adsorption of lactate dehydrogenase on ribosyl-linked NAD + discussed in the next section. By contrast, many other possible types of adjustment of the operating conditions--including, for example, pH and temperature changes--are more likely to disturb the bioaffinity and are less generally useful. Nevertheless, adjustment of the operating temperature has been found to be important in the perfection of at least one affinity chromatographic system. ~ In view of the temperature-dependence of at least some nonbiospecific
[8]
INTERFERENCE AND COMPLICATIONS
117
CNBr - activated Sepharose 4 B 1, 3-diaminopropan-2-ol ~ '~
OH I
~--NH--CH2--CH-- CH2--NH2 bromoacetylation OH
O
--NH --C H 2 - C H - - C H 2 - N H -
C-- CH2Br
diaminopropanol o
~
--NH--CH2--CH--CH2--NH --C --CH2--NH-- CH~-- C H - - C H 2 - - N H 2
FIG. 2. Structures and method of preparation of typical polar or hydrophilic spacer arms. Beaded agarose gel (Sepharose 4B, Pharmacia) is activated with CNBr in the usual manner [P. Cuatrecasas and C. B. Anfinsen, this series, Vol. 22, p. 345; P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970)]. (See also this volume [21 and [6].) The washed, activated gel is quickly mixed with an equal volume of an ice-cold 3 M solution of 1,3-diaminopropan-2-ol adjusted to pH 10, and the mixture is stirred gently at 4 ° for about 20 hours. The resulting derivatized gel (I) is washed with about 40 volumes of 0.2 M saline followed by water, and it is then bromoacetylated as described by Cuatrecasas (loc. cit). The bromoacetylated gel is mixed with an equal volume of 0.4 M 1,3-diaminopropan-2-ol in 0.1 M NaHCO:, buffer adjusted to pH 9, and the mixture is stirred gently at room temperature (ca. 15 °) for 3 days. The gel is washed with 0.1 M saline (about 40 volumes) and with distilled water (about 10 volumes), and any unsubstituted bromoacetyl groups remaining are blocked by incubation of the gel with 0.2M mercaptoethanol in 0.l M NaHCO~ buffer, pH 9 at 15 ° for 24 hours followed by washing as before. The process of bromoacetylation followed by addition of diaminopropanol may be repeated to yield even longer arms. The gels may be further derivatized by a number of the general methods described throughout this volume so as to provide suitable termini for attachment of the ligands. Data from P. O'Carra, S. Barry, and T. Griffin, unpublished. a d s o r p t i o n effects and p r o b a b l y of m a n y bioaffinities, it should be b o r n e in mind that an affinity system which works well at one t e m p e r a t u r e m a y be m u c h less successful, or m a y require f u r t h er o p e r a t i o n a l adjustments, if the o p e r a t i n g t e m p e r a t u r e is c h a n g e d ? ,24 In several cases of a d s o r p t i o n of enzymes on h y d r o p h o b i c spacer arms, salt c o n c e n t r a t i o n s even a b o v e 1 M did n o t h i n g to a m e l i o r a t e the interf e r en ce (see e.g., the e x a m p l e s of /3-galactosidase and alcohol d e h y d r o genase in the next s e c t i o n ) . A t t e m p t s to c o u n t e r a c t such severe h y d r o -
118
COUPLING REACTIONS AND GENERAL METHODOLOGY
[8]
phobic interference by addition of organic solvents or urea to the irrigant have met with little success, mainly because adequate concentrations of these additives are also capable of denaturing the enzymes. However, the alternative general approach--modification of the adsorbent, particularly the spacer a r m s - - h a s shown promise with a number of such "sticky" enzymes. The most successful tactic has been to incorporate polar groups, e.g., carbinol, amido and secondary amino groups, at regular intervals along the length of the spacer arms so that no extensive hydrophobic region remains. Prototype examples of such hydrophilic arms are shown in Fig. 2. Evidence of the effectiveness of such modified spacer arms in eliminating nonbiospecific adsorption is to be found in examples devoted to fl-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, and yeast alcohol dehydrogenase in the next section. Such hydrophilic spacer arms appear to eliminate nonbiospecific adsorption so completely that even the marginal level of such adsorption required as a reinforcing factor in compound affinity is also eliminated (cf., in next section, the examples in subsections on azo-linked NAD + and on the 6-1inked analogs of N A D + and A M P ) . Some Examples f l - G alactosidase
The affinity chromatographic system developed by Steers et al. 7 for fl-galactosidase employs a fl-thiogalactoside ligand attached to agarose via a spacer arm, as represented by structure (III). The method of elution is of the "deforming buffer" type and employs 0.1 M borate at pH 10. This affinity system promoted very strong adsorption of the enzyme even when the substitution level of the gel was as low as 0.6 mM, i.e., 0.6/zmole of ligand per milliliter of packed gel5 The long spacer arm of derivative (III) was found to be necessary for this strong adsorption, derivatives with shorter arms being ineffective. ~ This was interpreted purely in terms of elimination of steric hindrance, and these results have been widely quoted in support of the view that really long spacer arms may be necessary to eliminate steric hindrance. 7,l°-13,~-~ However, since the K~ value for the fl-thiogalactoside ligand is as high as 5 raM, r,'l there is an apparent wide discrepancy between the very weak retardation predicted by Eq. 1 (above) and the very strong adsorption observed with derivative ( I I I ) 2 , 9 Furthermore, satisfactory bioelution was not observed with this system2 ,7 "~P. Cuatrecasas, J. Biol. Chem. 245, 3059 (1970).
[8]
INTERFERENCE
AND COMPLICATIONS o
119
o
--NH--CH2-- CH2--CH~--NH--CHz--CH2--CH2--NH--C--CH2-- CH~--C --HN ~
S-~3-Galactoside
(III)
O O --NH--CH~--CH2 CHz--NH--CH~ CH2--CH2--NH--C--CH2--CH2--C--HN~---O-c~-GIucoside (iv)
~--NH--CH~
CH2--CH~--CH2--CH~--CH2--NH C--CH2--HN ~
(v) Nonbiospecific analogs of derivative (III) were thercfore prepared and studied. Derivative (IV) is an analog which differs from (III) in that the fl-galactoside ligand is rcplaced by an a-glucoside residue (for which fl-galactosidase should show little or no bioaffinity). Derivative (V) is a rcpresentative example of a number of "mock affinity gel" carrying no recognizable ligand at all. The chromatographic behavior of fl-galactosidase on dcdvativc (IV) was indistinguishable from its behavior on derivative (III) '~,ls and the enzyme was even more strongly adsorbed on the ligandless derivative (V) and on a numbcr of similar analogs. Elution could still be achieved with 0.1 M borate at pH 10, but only slowly and with much tailing2 ,'~ The enzyme was not eluted from any of the gels by biospecific countcrligands, i.e., soluble fl-galactosides, even when high salt concentrations were applied at the same time2 .s.'s The adsorption involved in this system therefore seems to be predominantly, attributable to nonbiospecific interactions involving the spacer arms. The hydrocarbon nature of these arms and the apparent ineffectiveness of high salt concentrations in counteracting the adsorption suggest that the interactions are of a hydrophobic nature. This seems to be confirmed by the fact that fl-galactosidase was not strongly adsorbed on gels carrying polar or hydrophilic spacer arms such as that of derivative (II) (Fig. 2). Ribonuclease A
The affinity gel developed by Wilchek and Gorecki 2G for ribonuclease A utilizes as adsorbent agarose-APUP, a uridine 2'(3'),5'-diphosphate ligand immobilized by attachment to agarose via an aminophenyl spacer group. The enzyme is satisfactorily adsorbed on this gel only when applied ~;M. Wilchek and M. Gorecki, Eur. J. Biochem. 11, 491 (1969). See also this volume [57].
120
COUPLING REACTIONS AND GENERAL METHODOLOGY
[8]
in dilute buffer at pH 5.2. In the original work elution was achieved with 0.2 M acetic acid, but elution can also be achieved by raising the pH to 7.5 at moderate ionic strength (0.1 to 0.2 M KCI) or by raising the ionic strength to a high level (0.5 to 1 M KC1) at pH 5.2. ~ The pH optimum of the enzyme is in the region of pH 7.0, and salt concentrations up to 0.2 M are not notably inhibitory. 2s It is therefore difficult to rationalize the adsorption and elution behavior satisfactorily in terms of pure bioaffinity. In view of the anionic nature of the ligand, due to its phosphate groups, the possibility that the adsorption may be complicated by nonbiospecific ion-exchange effects must be considered. This possibility is also suggested by the fact that the chromatographic behavior of bovine pancreatic ribonuclease A on simple anionic ion-exchange resins such as IRC-50 and CM-cellulose is in many respects similar to its behavior on the affinity gel; adsorption of the enzyme is strong at or near pH 5.2 and is weakened by raising the pH or by increasing the ionic strength. 2~,2'~ The enzyme may also be eluted--though much less satisfactorily--with aqueous acetic acid. z7 Difficulties have been reported by several workers who have used this affinity system in the purification of ribonucleases from sources other than beef pancreas. A ribonuclease from a myxomycete '~° and one of broad specificity from a shellfish source "~1 behaved quite differently from the bovine pancreatic enzyme; the purification achievable in these cases was not considered useful. Even pancreatic ribonuclease from other mammalian species behaved differently and modifications of the operating conditions had to be developed by a trial-and-error approach rather than by logic based on biospecificity. 3~ These difficulties are understandable if the adsorption and elution depend largely on gross electrostatic properties since these bear no necessary relationship to biospecificity. Adsorption of Lactate Dehydrogenase on Ribosyl-Linked N A D +
Lowe and Dean 17,'33 have described model affinity chromatographic studies of lactate dehydrogenase and other dehydrogenases on a "*~C. M a h o n and P. O'Carra, unpublished work. "°SF. M. Richards and H. W. Wyckoff, in "The Enzymes" (P. D. Boyer, ed.), 3rd Ed., Vol. IV, p. 647. Academic Press, New York (1971). " C . H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 200, 493 (1953). '~°D. R. Farr, H. Amster, and M. Horisberger, Arch. Microbiol. 85, 249 (1972). 31W. Murphy and D. B. Johnson, Comp. Biochem. Physiol. in press; S. Bree, D. B. Johffson, and M. P. Coughlan, Comp. Biochem. Physiol. in press. 32R. K. Wierenga, J. D. Huizinga, W. Gaastra, G. W. Welling, and J. J. Beintema, F E B S Lett. 31, 181 (1973). 33C. R. Lowe and P. D. G. Dean, Biochem. J. 133, 515 (1973).
[8]
INTERFERENCE AND COMPLICATIONS
121
number of immobilized N A D ~ preparations. One of these was prepared by direct coupling of N A D ~ to a CNBr-activated polysaccharide matrix and another by carbodiimide-promoted attachment of N A D + via the terminal carboxyl group of a caprylic acid-substituted polysaccharide matrix. It was initially assumed that the point,of attachment of the N A D ÷ in these derivatives was the 6-amino group of the adenine residue, 17 but it has since been established that attachment is mainly, if not entirely, through the ribosyl hydroxyl groups of the N A D + . 6,34 In the model studies of Lowe and Dean, 17,~'~ enzymes are applied in a buffer of low ionic strength and elution is achieved by a concentration gradient of KCI. These authors quantify affinity chromatographic effectiveness in terms of "binding" which is defined as the millimolar salt concentration needed to elute an enzyme. On the basis of this criterion both of the above-mentioned immobilized N A D ÷ derivatives were considered to be effective affinity systems for lactate dehydrogenase. However, kinetic studies show that the KC1 concentrations used for elution of this enzyme have little or no effect on the biospecific binding of soluble N A D ÷ to lactate dehydrogenase. ~ Moreover, at the low ionic strength of the starting buffer the adsorbed enzyme is not susceptible to bioelution by soluble N A D ~ added to the irrigant. This evidence suggests that the adsorption-elution behavior observed with these derivatives is dominated by salt-sensitive, nonbiospecific adsorption. Dean and Lowe 15 and Lowe et al. ~ have suggested that the CNBr activation step introduces ionic groups into the matrix which must be neutralized or eliminated before satisfactory results are obtainable. However the N A D + residue itself is ionic and would be expected to promote some ion-exchange adsorption at low ionic strength. This presumably dominates the chromatography, since at most only very weak bioaffinity can be detected with the ribosyl-linked N A D ÷ derivatives under discussion2 This is consistent with the detailed picture of the NAD~-binding site of lactate dehydrogenase which has recently emerged from the X-ray diffraction studies of Rossman and co-workers'~,~: both ribosyl residues of the N A D + molecule are buried in the binding crevice and are involved in the binding interactions of N A D ~ with the enzyme. Attachment of the N A D ÷ molecule ~' K. Mosbach, H. Guilford, R. Ohlsson, and M. Scott, Biochem. J. 127, 625 (1972). :~C. R. Lowe, M. J. Harvey, D. B. Craven, and P. D. G. Dean, Biochem. J. 133, 499 (1973). ~" M. G. Rossman, M. J. Adams, M. Buehner, G. C. Ford, M. L. Hackert, P. 1. Lentz, Jr., A. McPherson, Jr., R. W. Schevitz, and I. E. Smiley, Cold Spring Harbor Symp. Quant. Biol. 36, 179 (1972). '~M. J. Adams, M. Buchner, K. Chandrasekhar, G. C. Ford, M. L. Hackert, A. Liljas, M. G. Rossman, I. E. Smiley, W. S. Allison, J. Everse, N. O. Kaplan, and S. S. Taylor, Proc. Nat. Acad. Sci. U.S. 70, 1968 (1973).
122
COUPLING
REACTIONS
AND
GENERAL
METHODOLOGY
[8]
through either of these residues would therefore be expected to drastically decrease or abolish the bioaffinity of the resulting derivative. Azo-Linked
NAD +
Immobilized N A D + derivatives may also be prepared by incubation of N A D + with diazotized derivatives of matrix-spacer arm assemblies such as derivative ( V I ) . 6 The N A D + is thought to attach through the 8-position of the adenine residue. 6'~s"Immobilized N A D P + analogs can be similarly prepared, 6 and N A D + has been similarly attached via the hydrophilic spacer arms described above in the section on control of interference. 18 A group of such derivatives, ( V I ) to ( I X ) , have been used for affinity chromatographic studies of a range of NAD+-linked dehydrogenases, and these studies have provided useful insights into some of the complications that can be encountered in affinity chromatography. 6,1s // - - NH-- CH 2 - CH z - CH2- - CH z - - CH 2- - CH 2 - NH -- C ~ x
\\ ~-NH2
(vi)
--NH --CH2--CH2--CH2--CH2--CH2--CH2--NH --C -~<
~
N----N--NAD
(vii)
~--NH--CH2--CH2--CH2--CH~--CH2--CH2--NH--~~N~N--NADP + (vm)
OH
0 II
OH l
0 )I
#
\\
--NH--CH2--CH--CH2--NH--C--CH2--NH--CH2--CH--CH2--NH--C ~ - - N ~ N
NAD +
(LX)
Yeast ,41coholDehydrogenasefi,IsThis
enzyme was firmly adsorbed on
derivative ( V I I ) , but it could neither be eluted by addition of soluble N A D + to the irrigant nor by KC1 concentrations as high as 1 M. Addition of both N A D + and high salt concentrations to the irrigant simultaneously also failed to elute the enzyme. On the other hand, the enzyme showed no detectable affinity for the hydrophilic derivative ( I X ) . It thus seems likely that the adsorption on ( V I I ) is predominantly or entirely due to interac3s M . K . W e i b e l , H . H . W e e t a l l , a n d H . ] . B r i g h t , 44, 347 ( 1 9 7 1 ) .
Biochem. Biophys. Res. Commun.
[8]
INTERFERENCE AND COMPLICATIONS
123
tions with the hydrophobic spacer arm of this derivative. The enzyme is also adsorbed on a number of agarose derivatives carrying hydrophobic groups and no biospecific ligand. TM Glyceraldehyde-3-phosphate Dehydrogenase. '~,1~ The enzyme from rabbit muscle is adsorbed strongly on derivative (VII). The adsorption is not noticeably affected by high concentrations of KC1, and the enzyme is cleanly eluted by 2 mM NAD + if a salt concentration of at least 0.2 M is maintained in the irrigant. Under such conditions the enzyme is not adsorbed on either the ligandless analog ( V I ) or on the NADP + analog (VIII). These results indicate clearly the biospeeific nature of the adsorption of this enzyme on derivative (VII). However, the yield of the enzyme elutable with NAD ~ becomes progressively lower the longer the enzyme is left adsorbed on the gel before elution. This loss is far in excess of the loss of activity in control experiments when samples of the enzyme are put through equivalent procedures on a "blank gel" such as derivative (VI). The hypothesis that this time-dependent loss of activity is due to slow nonbiospecifie adsorption onto the hydrophobic spacer arm, after the enzyme has first bound biospecifically to the immobilized NAD + is supported by the results obtained when the spacer arm is replaced by a hydrophilic one as in derivative ( I X ) . The enzyme is just as strongly adsorbed on this derivative as on derivative ( V I I ) , but when it is eluted with NAD ÷, the yield is greater than 90% and does not decrease significantly even when the enzyme is left on the column for 1.5 hours, by which time the yield of NAD+-elutable enzyme from derivative (VII) has decreased to about 10%. Lactate Dehydrogenase. 6,1s When lactate dehydrogenase is chromatographed at low ionic strength, e.g., in 0.05 M potassium phosphate at pH 7.4, it is strongly adsorbed on derivative ( V I I ) , but is not bioelutable; it is similarly adsorbed onto the ligandless analog (VI). At low ionic strength, therefore, the adsorption is dominated by nonbiospecific adsorption attributable to the matrix-spacer arm assembly. Inclusion of 0.5 M KC1 abolishes such interfering adsorption, as evidenced by the lack of adsorption of the enzyme on derivatives (VI) and (VIII) under such conditions. However, at such a high salt concentration the enzyme remains only weakly retarded by derivative (VII). Since such salt concentrations do not interfere significantly with the binding of soluble NAD ÷ to lactate dehydrogenase, 6 this result suggests that, although the azo-linked NAD + derivative retains some bioaffinity for this enzyme, it is rather weak, presumably as a result of partial steric hindrance by the immobilization linkage. This seems to be confirmed by the fact that lactate dehydrogenase
124-
COUPLING
REACTIONS
AND
GENERAL
METHODOLOGY
[8]
o --NH --CH~
C H 2 - - C H 2 - - C H 2 - - CH2-- C H 2 - - N H - - C - - C H z S
N
O
N
C--NH O-
O-
l /O--CH,--O--Ip--o--I--o--CH, O. I HO
OH
HO
OH
(x)
i
o - - N H -- C H 2 - C H 2-- CIl2-- C H 2-- Cl£z-- C H 2- - N H -- C -- C H 2
N
N
O-
HO
OH
(xl) OH I
0
OH
I¢
--NH--CH2--CH--CH2--NH--C--
I
CH2--NH--CHz--CH--CH~--NH--CO--
CH 2 S
OCH2--O--~?O
HO
OH
(Xm
shows only very weak affinity for derivative ( I X ) at salt concentrations as low as 0.1 M. The inclusion of 0.2 M KC1 in the irrigating buffer was sufficient to largely eliminate the nonbiospecific adsorption of this enzyme on derivatives (VI) and (VIII). At this intermediate ionic strength, lactate dehydrogenase was strongly adsorbed on derivative (VII), but not on derivative ( I X ) , and could be cleanly eluted with the biospecific counterligands N A D H and 5'-AMP (a weak competitive inhibitor against NAD+). This seemingly clear-cut bioaffinity is of a much stronger nature than could be accounted for by bioaffinity alone, in view of the results at high ionic strength and those with the hydrophilic spacer arm. The discrepancy is most plausibly explained on the basis of the compound affinity concept discussed in a previous section.
[8]
INTERFERENCE AND COMPLICATIONS
125
Analogs o[ N A D ÷ and A M P Linked through the 6 Position o[ the Purine Residue ls,39
Derivatives (X) to ( X I I ) may be regarded as immobilized thio analogs of NAD + and 5'-AMP linked through the 6-position of the purine residue. They are easily prepared by reacting the 6-mercaptopurine analogs of the nucleotides with bromoacetyl-terminating matrix-arm assemblies. '~9 This linkage procedure was considered promising for affinity chromatography of certain NAD-linked dehydrogenases, including lactate dehydrogenase, because it was known from kinetic studies that derivatization at the 6-position does not drastically affect binding of NAD + to these enzymes. Mosbach et al. 34,4°,41 have synthesized 6-1inked derivatives by another method on the basis of the same rationale. Lactate de hydrogenase was strongly adsorbed on the 6-immobilized NAD + analog ( X ) ? 9 Bioelution with soluble N A D + was clear-cut if the salt concentration in the irrigant was maintained above 0.1 M and the affinity remained strong at high ionic strengths (e.g., 0.5 M KCI) indicating that the strong affinity of derivative (X) for this enzyme is probably pure bioaffinity. This contrasts with the behavior of the azo-linked derivative (VII) which binds lactate dehydrogenase strongly only at moderate ionic strengths, apparently with the help of nonbiospecific reinforcement (see above). 5'-AMP is a weak competitive inhibitor of lactate dehydrogenase (K~ = ca. 2 mM). At the substitution level of the batches of derivative (XI) prepared to date ( < 1 mM), only very weak biospecific retardation would be predicted from Eq. 1. In agreement with this, the enzyme was only marginally adsorbed on derivative ( X I ) at high ionic strength (0.5 M KC1). However, at low ionic strength the enzyme was strongly adsorbed on this derivative and the ionic strength could be adjusted to intermediate values (e.g., 0.1 M) such that the enzyme remained strongly adsorbed but could be cleanly eluted with biospecific counterligands, such as 5'AMP and NADH. On the hydrophilic derivative (XII) the enzyme remained only marginally adsorbed at such intermediate ionic strength demonstrating the positive contribution of the hydrophobic arm of derivative ( X I ) to the compound affinity. Malate dehydrogenase is the least "sticky" enzyme so far encountered in our work. The enzyme from beef heart is only marginally retarded by hydrophobic "mock affinity gels," such as derivative (VI), even at low ionic strengths. This enzyme displays no detectable affinity for the azo:'"S. Barry and P. O'Carra, FEBS Lett. 37, 134 (1973). '°R. Ohlsson, P. Brodelius, and K. Mosbach, FEBS Lett. 25, 234 (1972). ~1K. Mosbach, this volume [16].
126
COUPLINGREACTIONS AND GENERAL METHODOLOGY
[9]
linked N A D + derivative ( V I I ) but is strongly adsorbed on the 6-1inked N A D ÷ analog ( X ) at moderate ionic strengths (0.1-0.2 M ) and is cleanly bioelutable with N A D H . Malate dehydrogenase shows only very weak affinity for the 6-1inked A M P analog ( X I ) and, in contrast to lactate dehydrogenase, this affinity is not reinforced at moderate ionic strengths, presumably owing to the general lack of nonbiospecific interaction of this enzyme with hydrophobic spacer arms. 4~ ,5 p. O'Carra and S. Barry, unpublished work (1973).
[9] Hydrophobic
Chromatography
B y SHMUEL SHALTIEL
D e v e l o p m e n t of the Basic Principle In the course of our studies on enzymes involved in glycogen metabolism, we attempted to coat beaded agarose with glycogen and use it for affinity chromatography 1 of these enzymes. The procedure used for the preparation of the columns consisted of: (a) activation of the agarose with CNBr z and reaction with an a,,0-diaminoalkane to obtain an ,o-aminoalkyl agarose, ( b ) binding of CNBr-activated glycogen to the o,-aminoalkyl agarose. Two glycogen-coated agarose preparations which differed only in the length of the hydrocarbon chains bridging the ligand and the bead (Seph-C8-NH-glycogen 3 and Seph-C4-NH-glycogen) displayed unexpected differences in the retention of glycogen phosphorylase b. Whereas the column with 8-carbon-atom bridges adsorbed the enzyme, the column with 4-carbon-atom bridges did not even retard it. 4 Considering the large molecular dimensions of glycogen (Fig. 1 ), it did not seem likely that changing the length of the extensions from 8 to 4 carbon atoms would have such a dramatic effect on the retention power of the column. Rather, we were initially inclined to think that the column with the shorter side chains had failed to bind glycogen because of a reduced accessibility of the amino groups. This possibility was excluded by repeating the syn1p. Cuatrecasas, M. Wilchek, and C. B. Anfinsen, Proc. Nat. Acad. Sci. U.S. 61, 636 (1968). ~R. Ax6n, J. Porath, and S. Ernb~ick, Nature (London) 214, 1302 (1967). ~Seph-C~-NH-glycogen represents Sepharose 4B activated with CNBr, reacted With an ~,~-diaminoalkane n-carbon atoms long, then coupled with CNBr-activated glycogen. 4Z. Er-el, Y. Zaidenzaig, and S. Shaltiel, Biochem. Biophys. Res. Commun. 49, 383 (1972).