866
BIOCHEMICAL SOCIETY TRANSACTIONS
I acknowledge the Medical Research Council of Ireland for generous financial support and Professor Brian Leonard, Department of Pharmacology, University College, Galway, for many stimulating discussions.
Dolly, J. O., Dillon, A., Duffy, M. J. & Fottrell, P. F. (1971) Clin. Chim. Actu 31, 55-62 Donlon, J. & Fottrell, P. F. (1971) Clin. Chim. Acta 33, 345-350 Donlon, J. & Fottrell, P. F. (1972) Cornp. Biochem. Physiol. B 41, 181-193 Fogel, M. R. & Adibi, S. A. (1974) J. Lab. Clin. Med. 84, 327-333 OCuinn, G. & Fottrell, P. F. (1975) Biochem. Soc. Trans. 3, 1219-1221 OCuinn, G., Piggott, C. 0. & Fottrell, P. F. (1975) FEBS Lett. 53, 164-166 Peters, T. (1970) Gut 11,720-725
Assnity Chromatography of Biliverdin Reductase JANE WAIT and PADRAIG O’CARRA
Department of Biochemistry, University College, Galway, Ireland The first product of haem catabolism, the green pigment biliverdin, is converted rapidly in mammals into the familiar yellow bile pigment, bilirubin. The reductive conversion is catalysed by an NAD(P)H-dependent enzyme, biliverdin reductase, which is located chiefly in the liver and/or spleen of most mammals (Singleton & Laster, 1965; O’Carra et al., 1975; Colleran & O’Carra, 1976).
+
+
Biliverdin NADPH or NADH H+
-
Blllverdln
rsducfasc
+
Bilirubin NADP+ or NAD+
Efforts to purify this enzyme in native form have not been notably successful. The dficulties have been ascribed by O’Carra & Colleran (1971) to a marked lability of the enzyme towards adsorptive processes, which provide the most useful ‘conventional‘ purification methods for enzymes. Thus ion-exchange chromatography and adsorption chromatography [e.g. on Ca,(PO,), gel] yielded greatly destabilized enzymepreparations with altered kinetic properties. Tenhunen et al. (1970) used such chromatography to achieve a 52-fold purification of biliverdin reductase, but the purified enzyme was highly unstable and its reported kinetic properties differ considerably from those described by O’Carra & Colleran (1971) for the native enzyme. Eschewing adsorptive purification steps to avoid destabilization, O’Carra & Colleran (1971) were able to achieve an overall purification of only 15-fold [by using (NH&S04 fractionation followed by gel filtration] and we have been unable to improve on this by using ‘conventional’ purification techniques. The chromatographic destabilization does not appear to be caused by loss of a ‘stabilizingfactor’(Colleran, 1971), but rather it seems to arise through the gross physicochemical interaction of the enzyme with the adsorbents. This seems unavoidable in ‘conventional’ chromatographic procedures, but in affinity chromatography the adsorption of the enzyme is based on biospecific interaction of the active site with immobilized substrate analogues rather than on gross physicochemical interactions. We therefore investigated the potential of affinity chromatography in the purification of biliverdin reductase. Although technical difficulties have prevented us from exploiting the full potential of the approach, we have nonetheless been able to improve the purification factor to 250-fold without destabilizing the enzyme or altering its kinetic properties. Potential ligands for affinity chromatography of biliverdin reductase include biliverdin and the nicotinamide nucleotide cofactors. (The bilirubin product shown little affinityfor the enzyme,which catalysesan essentially irreversiblereaction.) Biliverdin is a highly specific substrate for this enzyme and also has a very high affinity for it (K, = 0 . 2 with ~ ~ NADPH as co-substrate) (Colleran & O’Carra, 1970). It would therefore have been the most suitable ligand on which to base an affinity-chromatographysystem. Unfortunately our efforts to prepare a suitable immobilized analogue of biliverdin have
1976
564th MEETING, DUBLIN
Agarosc -NH--(CHJe
867
-CH-
Fig. 1. Immobilized NADP+ derivative used The NADP+ is linked via the azo linkage through the 8 position of the adenine group. The gel was prepared by converting agarose 4B (Pharmacia, UppsaIa, Sweden) into the p-aminobenzamidohexyl derivative by the procedures of Cuatrecasas (1970) and Barry & O’Carra (1973). This was then diazotized and linked with NADP+ by the procedure of Barry & OCarra (1973) for similar immobilization of NAD+
I
Effluent volume (column-volume units) Fig. 2. Elution profires of bilverdin reductase chromatographed on the immobilized NADP+ (Fig. 1) The enzyme was extracted from bovine spleen and pre-pursed by the procedure of O’Carra & Colleran (1971). The irrigating buffer throughout was 0.1M-potassium phosphate buffer, pH7.4, containing O . ~ M - K C to~ minimize non-biospecific interaction. Further increases in the KCl concentration had no noticeable effect on the results, nor did it elute the adsorbed enzyme. Additions of soluble NADPH (a; 1m)or NADH ( 6 ; IOmM) were made to the irrigating buffer (indicated by arrows) to effect competitive elution of the enzyme, which was assayed as descibed by Colleran (1971). The point of application was at 0 column volumes.
so far been unsuccessful. Immobilization through the side-chain carboxylate groups is comparatively easy chemically, but these carboxylate groups are important in the affnity of biliverdin for the enzyme active site. The well-known chemical lability of biliverdin has defeated our attempts to immobilize it successfully through other side-chain positions. Both NADPH and NADH act as substrates for biliverdin reductase, but the affinity for NADPH (K,,,=~,uM)is an order of magnitude higher than that for NADH ( K , = 5 0 0 ~ (Colleran ~ ) & O’Carra, 1970). Their products, NADP+ and NAD+, also bind to the active site as competitive inhibitors, with a similar difference in affinities ( K , for NADP+ = 4pM; K, for NAD+ = 6 5 0 , ~ ~ Both ). NADP+ and NAD+ were immobilized successfully on a Sepharose 4B gel derivative by following the azo-linkage procedure described by Barry & O’Carra (1973) (Fig. 1). When partially purified biliverdin reductase was applied to columns of these affinity gels it displayed differential affinities for the two immobilized cofactors consistent with their inhibition constants. The immobilized NAD+gel promoted little adsorption, whereas theenzyme was strongly VOl. 4
868
BIOCHEMICAL SOCIETY TRANSACTIONS
adsorbed on the immobilized NADP+ gel (Fig. 2). A similar differential effect was evident in the relative abilities of NADPH and NADH to elute the enzyme from the NADP+ gel. As illustrated in Fig. 2, NADPH eluted the enzyme cleanly (showing that it competesfavourably with the immobilized NADP+),but NADH, even at a 10-fold higher concentration, caused only slow elution of the enzyme, consistent with its lower affinity. The fact that biliverdin reductase can be eluted from the immobilized NADP+ with soluble NADH shows that there is competition between NADP+ and NADH for the same binding site on the enzyme and confirms that the NADPH- and NADHdependent activities are associated with the one enzyme (cf. Colleran 8c O’Carra, 1970, 1976). As regards purification of the enzyme, elution with NADH would be theoretically preferable to elution with:NADPH,makingthechromatography bothNADP-and NADspecific, but in practice elution with NADH leads to severe tailing and dilution of the eluted enzyme, thus making it impractical as a purification step. The best purification to date was achieved by using NADP+ as immobilized ligand and NADPH as eluting counter-ligand. An overall purification factor of 250-fold is achieved as a routine. There is, unfortunately, a tendency for the enzyme, once adsorbed by the immobilized NADP+ to interact non-specifically with other elements of the gel, probably the spacer arm (see Fig. 1). Such non-specific interaction seems to lead to inactivation and low recoveries of the enzyme. The problem seems to be analogous to that described for glyceraldehyde 3-phosphate dehydrogenase by Barry & O’Carra (1973). It can be minimized by inclusion of a high concentration of a neutral salt in the irrigating buffer (0.5~-KC1was used as a routine in our work). This does not overcome the problem completely and variable losses of enzyme activity were encountered during chromatography. Nonetheless the eluted enzyme is quite stable and seemingly native, in contrast with preparations subjected to conventional adsorption-chromatographic procedures. We thank the Medical Research Council of Ireland for a grant-in-aid and the Irish Department of Education for a Research Maintenance Grant to J. W. Barry, S. & O’Carra, P. (1973) Biochem. J. 135, 595-607 Colleran, E. (1971) Ph.D. Thesis, National University of Ireland Colleran, E. & O’Carra, P. (1970) Biochem. J . 119, 1 6 ~ - 1 7 ~ Colleran, E. & O’Carra, P. (1976) in Chemistry and Physiology of Bile Pigments (Berk, P. D. & Berlin, N. I., eds.), U.S.A. Government Printing Office,Washington, DC, in the press Cuatrecasas, P. (1970) J. Biof. Chem. 245, 3059-3065 O’Carra, P. & Colleran, E. (1971) Biuchern. J. 125, l l 0 p OCarra, P., Colleran, E. & Delaney, M. (1975) in Metabolism and Chemisfry of Bilirubin and Related Tefrapyrrols (Bakken, A. F. & Fog, J., eds.), pp. 124126, Pediatric Research Institute, Oslo Singleton, J. W. & Laster, L. (1965) J. Biol. Chem. 240,4780-4789 Tenhunen, R., Ross, M. E., Marver, H. S. & Schmid, R. (1970) Biochemistry 9,298-303
Steroid Catabolism in Choline-Deficient Rats MARY E. BEARY and JOSEPH RAFTER Department of Biochemistry, University College, Dublin 4, Ireland
Choline, as phosphatidylcholine and sphingomyelin, constitutes a major structural component of the membranes of liver cells. Feeding with a choline-deficientdiet has been shown to result in structural and functional alterations in these membranes (Lombardi, 1971) as well as in the activity of membrane-bound enzymes having a functional requirement for phospholipids (Leelavathi et al., 1974). The ability of the liver to metabolize steroid hormones is dependent on enzyme systems bound to the endoplasmic reticulum. Evidence has been presented that 1976
BIOCHEMICAL SOCIETY TRANSACTIONS
I acknowledge the Medical Research Council of Ireland for generous financial support and Professor Brian Leonard, Department of Pharmacology, University College, Galway, for many stimulating discussions.
Dolly, J. O., Dillon, A., Duffy, M. J. & Fottrell, P. F. (1971) Clin. Chim. Actu 31, 55-62 Donlon, J. & Fottrell, P. F. (1971) Clin. Chim. Acta 33, 345-350 Donlon, J. & Fottrell, P. F. (1972) Cornp. Biochem. Physiol. B 41, 181-193 Fogel, M. R. & Adibi, S. A. (1974) J. Lab. Clin. Med. 84, 327-333 OCuinn, G. & Fottrell, P. F. (1975) Biochem. Soc. Trans. 3, 1219-1221 OCuinn, G., Piggott, C. 0. & Fottrell, P. F. (1975) FEBS Lett. 53, 164-166 Peters, T. (1970) Gut 11,720-725
Assnity Chromatography of Biliverdin Reductase JANE WAIT and PADRAIG O’CARRA
Department of Biochemistry, University College, Galway, Ireland The first product of haem catabolism, the green pigment biliverdin, is converted rapidly in mammals into the familiar yellow bile pigment, bilirubin. The reductive conversion is catalysed by an NAD(P)H-dependent enzyme, biliverdin reductase, which is located chiefly in the liver and/or spleen of most mammals (Singleton & Laster, 1965; O’Carra et al., 1975; Colleran & O’Carra, 1976).
+
+
Biliverdin NADPH or NADH H+
-
Blllverdln
rsducfasc
+
Bilirubin NADP+ or NAD+
Efforts to purify this enzyme in native form have not been notably successful. The dficulties have been ascribed by O’Carra & Colleran (1971) to a marked lability of the enzyme towards adsorptive processes, which provide the most useful ‘conventional‘ purification methods for enzymes. Thus ion-exchange chromatography and adsorption chromatography [e.g. on Ca,(PO,), gel] yielded greatly destabilized enzymepreparations with altered kinetic properties. Tenhunen et al. (1970) used such chromatography to achieve a 52-fold purification of biliverdin reductase, but the purified enzyme was highly unstable and its reported kinetic properties differ considerably from those described by O’Carra & Colleran (1971) for the native enzyme. Eschewing adsorptive purification steps to avoid destabilization, O’Carra & Colleran (1971) were able to achieve an overall purification of only 15-fold [by using (NH&S04 fractionation followed by gel filtration] and we have been unable to improve on this by using ‘conventional’ purification techniques. The chromatographic destabilization does not appear to be caused by loss of a ‘stabilizingfactor’(Colleran, 1971), but rather it seems to arise through the gross physicochemical interaction of the enzyme with the adsorbents. This seems unavoidable in ‘conventional’ chromatographic procedures, but in affinity chromatography the adsorption of the enzyme is based on biospecific interaction of the active site with immobilized substrate analogues rather than on gross physicochemical interactions. We therefore investigated the potential of affinity chromatography in the purification of biliverdin reductase. Although technical difficulties have prevented us from exploiting the full potential of the approach, we have nonetheless been able to improve the purification factor to 250-fold without destabilizing the enzyme or altering its kinetic properties. Potential ligands for affinity chromatography of biliverdin reductase include biliverdin and the nicotinamide nucleotide cofactors. (The bilirubin product shown little affinityfor the enzyme,which catalysesan essentially irreversiblereaction.) Biliverdin is a highly specific substrate for this enzyme and also has a very high affinity for it (K, = 0 . 2 with ~ ~ NADPH as co-substrate) (Colleran & O’Carra, 1970). It would therefore have been the most suitable ligand on which to base an affinity-chromatographysystem. Unfortunately our efforts to prepare a suitable immobilized analogue of biliverdin have
1976
564th MEETING, DUBLIN
Agarosc -NH--(CHJe
867
-CH-
Fig. 1. Immobilized NADP+ derivative used The NADP+ is linked via the azo linkage through the 8 position of the adenine group. The gel was prepared by converting agarose 4B (Pharmacia, UppsaIa, Sweden) into the p-aminobenzamidohexyl derivative by the procedures of Cuatrecasas (1970) and Barry & O’Carra (1973). This was then diazotized and linked with NADP+ by the procedure of Barry & OCarra (1973) for similar immobilization of NAD+
I
Effluent volume (column-volume units) Fig. 2. Elution profires of bilverdin reductase chromatographed on the immobilized NADP+ (Fig. 1) The enzyme was extracted from bovine spleen and pre-pursed by the procedure of O’Carra & Colleran (1971). The irrigating buffer throughout was 0.1M-potassium phosphate buffer, pH7.4, containing O . ~ M - K C to~ minimize non-biospecific interaction. Further increases in the KCl concentration had no noticeable effect on the results, nor did it elute the adsorbed enzyme. Additions of soluble NADPH (a; 1m)or NADH ( 6 ; IOmM) were made to the irrigating buffer (indicated by arrows) to effect competitive elution of the enzyme, which was assayed as descibed by Colleran (1971). The point of application was at 0 column volumes.
so far been unsuccessful. Immobilization through the side-chain carboxylate groups is comparatively easy chemically, but these carboxylate groups are important in the affnity of biliverdin for the enzyme active site. The well-known chemical lability of biliverdin has defeated our attempts to immobilize it successfully through other side-chain positions. Both NADPH and NADH act as substrates for biliverdin reductase, but the affinity for NADPH (K,,,=~,uM)is an order of magnitude higher than that for NADH ( K , = 5 0 0 ~ (Colleran ~ ) & O’Carra, 1970). Their products, NADP+ and NAD+, also bind to the active site as competitive inhibitors, with a similar difference in affinities ( K , for NADP+ = 4pM; K, for NAD+ = 6 5 0 , ~ ~ Both ). NADP+ and NAD+ were immobilized successfully on a Sepharose 4B gel derivative by following the azo-linkage procedure described by Barry & O’Carra (1973) (Fig. 1). When partially purified biliverdin reductase was applied to columns of these affinity gels it displayed differential affinities for the two immobilized cofactors consistent with their inhibition constants. The immobilized NAD+gel promoted little adsorption, whereas theenzyme was strongly VOl. 4
868
BIOCHEMICAL SOCIETY TRANSACTIONS
adsorbed on the immobilized NADP+ gel (Fig. 2). A similar differential effect was evident in the relative abilities of NADPH and NADH to elute the enzyme from the NADP+ gel. As illustrated in Fig. 2, NADPH eluted the enzyme cleanly (showing that it competesfavourably with the immobilized NADP+),but NADH, even at a 10-fold higher concentration, caused only slow elution of the enzyme, consistent with its lower affinity. The fact that biliverdin reductase can be eluted from the immobilized NADP+ with soluble NADH shows that there is competition between NADP+ and NADH for the same binding site on the enzyme and confirms that the NADPH- and NADHdependent activities are associated with the one enzyme (cf. Colleran 8c O’Carra, 1970, 1976). As regards purification of the enzyme, elution with NADH would be theoretically preferable to elution with:NADPH,makingthechromatography bothNADP-and NADspecific, but in practice elution with NADH leads to severe tailing and dilution of the eluted enzyme, thus making it impractical as a purification step. The best purification to date was achieved by using NADP+ as immobilized ligand and NADPH as eluting counter-ligand. An overall purification factor of 250-fold is achieved as a routine. There is, unfortunately, a tendency for the enzyme, once adsorbed by the immobilized NADP+ to interact non-specifically with other elements of the gel, probably the spacer arm (see Fig. 1). Such non-specific interaction seems to lead to inactivation and low recoveries of the enzyme. The problem seems to be analogous to that described for glyceraldehyde 3-phosphate dehydrogenase by Barry & O’Carra (1973). It can be minimized by inclusion of a high concentration of a neutral salt in the irrigating buffer (0.5~-KC1was used as a routine in our work). This does not overcome the problem completely and variable losses of enzyme activity were encountered during chromatography. Nonetheless the eluted enzyme is quite stable and seemingly native, in contrast with preparations subjected to conventional adsorption-chromatographic procedures. We thank the Medical Research Council of Ireland for a grant-in-aid and the Irish Department of Education for a Research Maintenance Grant to J. W. Barry, S. & O’Carra, P. (1973) Biochem. J. 135, 595-607 Colleran, E. (1971) Ph.D. Thesis, National University of Ireland Colleran, E. & O’Carra, P. (1970) Biochem. J . 119, 1 6 ~ - 1 7 ~ Colleran, E. & O’Carra, P. (1976) in Chemistry and Physiology of Bile Pigments (Berk, P. D. & Berlin, N. I., eds.), U.S.A. Government Printing Office,Washington, DC, in the press Cuatrecasas, P. (1970) J. Biof. Chem. 245, 3059-3065 O’Carra, P. & Colleran, E. (1971) Biuchern. J. 125, l l 0 p OCarra, P., Colleran, E. & Delaney, M. (1975) in Metabolism and Chemisfry of Bilirubin and Related Tefrapyrrols (Bakken, A. F. & Fog, J., eds.), pp. 124126, Pediatric Research Institute, Oslo Singleton, J. W. & Laster, L. (1965) J. Biol. Chem. 240,4780-4789 Tenhunen, R., Ross, M. E., Marver, H. S. & Schmid, R. (1970) Biochemistry 9,298-303
Steroid Catabolism in Choline-Deficient Rats MARY E. BEARY and JOSEPH RAFTER Department of Biochemistry, University College, Dublin 4, Ireland
Choline, as phosphatidylcholine and sphingomyelin, constitutes a major structural component of the membranes of liver cells. Feeding with a choline-deficientdiet has been shown to result in structural and functional alterations in these membranes (Lombardi, 1971) as well as in the activity of membrane-bound enzymes having a functional requirement for phospholipids (Leelavathi et al., 1974). The ability of the liver to metabolize steroid hormones is dependent on enzyme systems bound to the endoplasmic reticulum. Evidence has been presented that 1976
