The Determination of Dissociation Constants by Affinity Chromatography on an Immobilized Adenosine Monophosphate Analogue. PETER BRODELIUS and ...
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BIOCHEMICAL SOCIETY T R A N S A a I O N S
Lowe, C.R., Harvey, M. J. &Dean, P. D. G. (19746) Eur. J. Biochem. 42, 1-6 Nishikawa, A. H., Bailon, P. & Ramel, A. H. (1974) in Immobilised Biochemicals and Afinity Chromatography (Dunlap, R. B., ed.), pp. 33-42, Plenum Press, New York
The Determination of Dissociation Constants by Affinity Chromatography on an Immobilized Adenosine Monophosphate Analogue PETER BRODELIUS and KLAUS MOSBACH Biochemical Division, Chemical Centre, University of Lurid, P.O. Box 740, S-22007 Lund 7, Sweden Within a few years affinity chromatography has become a widely used technique for the separation and purification of enzymes. The development and advantages of general ligands in affinity chromatography have previously been reported (Mosbach et al., 1972; Ohlsson et al., 1972). Thus Sepharose-bound N6-(6-aminohexyl)-AMP has been used for the separation and purification of NAD+-dependent enzymes (Ohlsson et al., 1972, Lowe et al., 1974) as well as for the separation of the isoenzymes of lactate dehydrogenase (Brodelius & Mosbach, 1973). The method has, however, been used almost exclusively for the purification of enzymes. It is also possible to use the method for kinetic investigations. Thus the formation of ternary complexes with a number of dehydrogenases (Ohlsson et al., 1972) and the order of binding of substrates to lactate dehydrogenase (O’Carra & Barry, 1972) has been indicated. The five isoenzymes of bovine lactate dehydrogenase were resolved with a gradient of NADH into five distinct peaks by affinity chromatography on a column containing the Sepharose-bound AMP analogue. The separation was interpreted as reflecting the differences in the dissociation constant (Kdlss.)for the enzyme-NADH binary complex. In the present investigation a number of lactate dehydrogenases from different sources whose Kdlss. values were known from the literature were applied to such columns of Sepharose-bound AMP analogue and subsequently eluted with linear gradients of NADH. Fig. 1 shows a direct proportionality between these Kdlar. values and the eluting concentration of NADH. This linearity indicates that the other factor involved in affinity chromatography, i.e. the Kdiss.for the complex between enzyme and immobilized ligand, is in this case of minor importance for the elution process. The
I
0
0. I
0.2
0.3
-1
0.4
Eluting concentrationsof NADH (mM) Fig. 1. Dissociation constant for the binary complex between enzyme and NADH as a function of eluting concentration of NA D H The Kdiss.values were taken from the literature and are given in Table 1. 1974
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Table 1. Estimation of Kdlu. for the binary complexes between NADH and a number of lactate dehydrogenasesby affinity chromatography on an immobilized AMP analogue The KdLu.values were estimated from the eluting concentration of NADH by interpolation in Fig. 1. Eluting concentration Estimated of NADH (m) Kdiss. Literature value Isoenzyme Bovine H.,,crystalline
0.043
0.38
Bovine M4, crystalline
0.190
2.2
Pig H.,,crystalline
0.048
0.47
Pig H,, crude
0.047
0.46
Pig M4, crystalline
0.300
3.6
Rabbit M4, crystalline
0.255
3.1
Rabbit M4, crude
0.263
3.2
Chicken &,crystalline Chicken H4, crude Rat M4, crude
0.070 0.069 0.200
0.70 0.70 2.4
0.39 (Anderson & Weber, 1965) 2.0 (Stinson & Holbrook, 1973) 0.53 (Stinson & Holbrook, 1973) 0.53 (Stinson & Holbrook, 1973) 3.7 (Stinson & Holbrook, 1973) 3.2 (Fromm, 1963) 3.2 (Fromm, 1963)
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inhibition constant (K,) of the free AMP analogue for both bovine H4and M4isoenzymes for pig H4 isoenzyme (C. R. Lowe, personal (Brodelius & Mosbach, 1973) and its KdjsL). communication) has been shown to be in the order of 1 0 - 4 ~It. is likely that lactate dehydrogenases from other sources have Kdiss.values of a similar order and if the immobilization of the ligand does not alter this constant to any great extent, the elution pattern should be more sensitive to differences in the Kdiss.value for the enzyme-NADH complex, which would then be approximately 100 times lower, than to differences in the constant for the enzyme-immobilized ligand complex. A number of lactate dehydrogenases, including some, whose Kdiss.values were unknown to us, were applied to such affinity columns and subsequently eluted with gradients of NADH. By determining the eluting concentration of NADH and by interpolating in Fig. 1, the dissociation constants for the binary complexes between enzyme and NADH could be estimated. The results are summarized in Table I. It is noteworthy that no difference in the Kdiss.values were observed when crystalline and crude preparations were used. This is perhaps not surprising since the amount of enzyme applied to the column was well within its capacity. It is likely that other proteins in the crude preparation, which also bind to the column, do not influence the elution pattern. Conventional methods for the determination of dissociation constants require not only a pure enzyme but also a homogeneous isoenzyme. A distinct advantage of the present method is thus that a crude preparation, even a mixture of isoenzymes, can be used and obviates the necessity for pure samples. Further, the method is very rapid and requires only a very small amount of enzyme for each determination. In this case 1 unit (pmol/min) of enzyme was applied to a column containing lml of packed gel (140,umol of nucleotide/g of dry polymer) and once the affinity column was calibrated it was possible to estimate Kdiss.within a couple of hours, starting with a raw extract. The method is also presumably applicable to other enzymes that exploit AMP as a general ligand in affiity chromatography. VOl. 2
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BIOCHEMICAL SOCIETY TRANSACTIONS
Anderson, S. R. & Weber, G. (1965)Biochemistry 4,1948-1957 Brodelius, P. & Mosbach, K. (1973)FEBS Lett. 36,223-226 F r o m , H.J. (1963)J. Biol. Chem. 238,2938-2944 Lowe, C . R.,Harvey, M. J. &Dean, P. D.G.(1974)Eur. J. Biochem. 41,347-351 Mosbach, K.,Guilford, H., Ohlsson, R. & Scott, M. (1972)Biochem. J. 127,625-631 OCarra, P. & Barry, S. (1972)FEBS Lett. 21,281-285 Ohlsson, R.,Brodelius, P. & Mosbach, K. (1972)FEBS Lett. 25, 234238 Stinson, R. A. & Holbrook, J. J. (1973)Biochem. J. 131, 719-728
Affinity Chromatography Applied to the Purification of Rat Hepatic Glucokinase MICHAEL J. HOLROYDE and IAN P. TRAYER Department of Biochemistry, University of Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. The phosphorylation of glucose by hepatic glucokinase(EC 2.7.1.2) is an obvious point at which to regulate glucose metabolism in animals. It has not been possible to fully investigate the physiological control and mode of action of this enzyme because of the difficulties involved in preparing it by published methods (Parry & Walker, 1966; Pilkis, 1972). Such studies, as well as a precise kinetic analysis, require the availability of a homogeneous product, preferably obtained through a simple and efficient preparative procedure. A previous communication (Chesher et al., 1973) described a method for obtaining a highly pursed enzyme preparation direct from rat liver extracts by affinity chromatography on an agaros~N-(6-aminohexanoyl)-2-am~no-2-deoxy-~-glucopyranose matrix. Subsequent work has established that this method is much more effective in large-scalepreparations of theenzymeif it is preceded by an initial purification of the liver extracts on DEAE-cellulose. The glucokinase activity is adsorbed, in a batchwise procedure, on to the DEAE-cellulose at low ionic strength and eluted by increasing the KCI concentration. The enzyme, which is obtained in quantitative yield, can then be purified by repeated affinity chromatography. In the process of scaling-upthis affinity method, so that large numbers of rat livers can be dealt with, it became apparent that careful control of the ligand concentration on the gel was essential for its efficient operation. N-(6-Aminohexanoyl)-2-amino-2-deoxy-~glucopyranose is a competitive inhibitor of glucokinasewith respect to glucose, having a K , of 0.75~1~ Since . this glucosamine derivative has a relatively high K,for the enzyme, it is crucial in evaluating the best operating conditions for this affinity-chromatography step to obtain quantitative information on the interactions between this immobilized ligand, the enzyme and the specific displacing solute, glucose. At low ligand concentrations on the column, the enzyme activity ‘leaks’ from the column in increasing amounts before the inclusion of glucose in the developing buffer. The glucoseeluted fraction, however, possesses a very high specific activity. Increasing the ligand concentration caused the glucokinaseto be bound quantitatively and necessitated higher concentrations of glucose to effect its elution. At very high ligand concentrations, no enzyme was eluted by up to 1M-glucose. As the ligand concentrationwas increased, however, this resulted in binding of other proteins with a concomitant decrease in the purification achieved by the column. The ligand concentration is known to be crucial in the separation of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) from glucokinase by the immobilized glucosamine derivative. Thus for optimal use of this technique it is necessary to carefully control the amount of ligand coupled to the agarose matrix. The strength of binding of the enzyme and its relation to ligand concentration can best be explained on the basis of simple equilibria. This type of phenomenon has been noticed by workers for other enzymes (Harvey et al., 1974). Certain ADP-agarose conjugates, notably 8-(6-aminohexyl)amino-ADPand N6(6-aminohexyl)-ADP (Trayer et al., 1974), have also proved very effective affinity-
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