442
reviews
Designerdyes:‘biomimetic’ s for the purificationof pharmaceutical proteinsby affinitychromatography Christopher R. Lowe, Steven J. Burton, Nicolas P. Burton, Wendy K. Alder-ton, Jennifer M. Pit-k and Janette A. Thomas_ Affinity chromatography has been extensively refined over the past few years to meet the more stringent criieria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in processscale protein purification. Exploitation of sophisticated molecular modelling kchniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective ‘bicmimetic’ ligands for the target proteins.
Few of the recent advances in biotechnology would have been commercially viable without &cient methods of protein purification. Substantia; worldwide markets exist for highly purified monoclonal antibodies, human growth factors, cytokines, hormones, blood clotting factors and fibtinolytics, erythropoietin, vacciiies and diagnostic en,zymes. The isolation and purification of such proteins;&e the most critical steps in he delivery of phannac&tical products with full biological activity and which meet the specifications for clinical applications. All steps in the production of pharmaceutical proteins must be in compliance with current Good Manufacturing Practices (GMP), regulated by the Food and Drug Administration (FDA) in the USA and by similar agencies elsewhere, and must ensure that the final product is i;llly active and unadulterated. Thus, biological molecules isolated from nattiral sources, or expressed from recombinant DNA systems, must be shown to have acceptably low levels of biologically active contaminants such as DNA ((3Opg per dose) and other macromolecules, viruses, pyrogen (~300 endotoxin units per dose) and ‘leachates’ Tom the separation media. Not surprisingl~~, therefore, the development of gentle, but effective, purification processes which yield fully active pharmaceutical proteins presents new challenges to the process engineer.
AfEnity chromatography In general, purification processes should be designed to produce the highest purity product at the highest attainable yield, since every percent loss of yield that occurs in the downstream processing is multiplied by all the manufacturing costs that came before’. Consequently, an eccnomic process may be contingent solely on thz effectiveness of the purification process that has been developed. Affinity techniques such as affinirr chromarography harness molecular recognition encountered between biological macromolecules and complementary ligands such as enzyme substrates, inhibitors, effecters, coenzymes, hormones, antigens, nucleic acids and sugars’J. tinity technology has been extensively developed over the past few years in response to greater demands being Imposed by the increased production of commercially valuable recombinant therapeutic proteins and the associated quality criteria. These more stringent requirements ofscale, yield and product purity have led to a critical reappraisal of many of the early ideas of affinity chromatographyl. Thus, the qualities of the support matrix, the activation and coupling chemistry, the selection and stability of the ligand and the purification format itself have all been re-examined in the light of these new requirements. In particular, the ligand selected for immobilizaticn has been scrutinized closely in recent yea&. A fimd;lmental advantage of affinity techniques is their predictive and rational character, since the ligandselected is designed to interact specificaliy with the protein to be purified. In principle, this should simplie process design and make the selective adsorption step much less sensitive to the
-ll~___l___ TIBTECH DECEMBER
1992 NO1 10)
0
1992. Elsewer Science Publishers Ltd (UK)
443 reviews Table 1. Comparisonof immobilizedIbiomimetic’dyes wilh conventionalaffinity adsorbents
composition of the fecdstork. However, in spite of over 20 years ofempirical development, the technique has only recently begun to find favour for the purification of high-value bioproducts. This is probably due, in part, to the inherent cost and lability of the ligands used, in part, to the troublesome problems of non-specific adsorption and fouling and, in part, to the difficulties associated with the sterilization and sanitization of relatively unstable adsorbents. Despite these limitations, affinity chromatography is making inroads into large-scale protein purification6~7. This review describes new developments in the design of selective and stable ligands for affinity chromatography which should alleviate some of the previous deficiencies of the technique and further establish it as a routine tool in process-scale protein purification.
Criterion
Immobilized ‘biomimatic’dyes
Conventional biospecilfic adsorbents
cost
Inexpensive,
Can be expensive
Stability
Chemidly and biologically stable
Tend to be labile
Specificity
Moderate
fvloderate to high
Synthesis of adsorbents
Facile, often onestep reaction
Often lengthy, synthetic route and/or toxic activation reagents
Capacity
High (>lQ% ligand utiiization)
Low (O.Ol-10% ligand utilization)
Tikmimetic ligtmds A&&y adsorbents created with naturally occurring
Scale-up potential
Virtually limitless
Very limited
biological hgands rend to be expensive to produce since the ligands themselves are chemically and biologically labile and are difhcult to immobilize in high yield with retention of activit@s. However, the exploitation of ‘pseudo- ’CT‘hiomimetic’ ligands in
Reusability
Very high
LOW
Sterilizability
High
Mostly low
place of their natural counterparts as ligands for affinity chromatography offers the prospect of circumventing many of these difficulties. The commonest class of ‘biomimetic’ &and is the reactive textile dyes which originate from the serendipitous discovery that the dye component of blue dextran, Cibacron Blue F3G-A, bound to pyruvate kinase during gel filtration9. This chance observation aroused interest in the dye, and subsequently !ed to the putiiCication of many proteins (including aibumin and other blood proteins, coenzyme-dependent enzymes, decarboxylases, glycolytic enzymes, hydrolases, lyases, nucleases, polymerases, synthetases and transferases)3Jt-33 on immobilized Cibacron Blue FSG-A affinity adsorbents. The use of immobiiized textile dyes offers significant advantages over conventionaI affinity adsorbents (Table 1). They are inexpensive, commodity chemicals, readily coupled to a variety of support matrices via their reactive groups and resistant to biological and chemica! degradation. Immobilized dye adsorbents display high capacities for their complementary protein and thus have considerable scale-up potential. The mechanism of binding of Cibacron Blue F3G-A to proteins is still unclear; originally, it was thought that the anthraquinone ring (ring A in Fig. 1) and the bridging diaminobenzene sulphonate ring (ringl? in Fig. 1) bore superficial similarity to the structure of ATPI*. It was later proposed that Cibacron Blue F3G-A might be a conformational analogue of the entire NAD’ molecule and thus might prove to be a diagnostic probe for the dinucleotide binding domain’s. However, this idea has been largely discredited since many proteins lacking this structural feature bind the dye, whilst some with the fold do not bind. Furthermore, kinetic and chromatographic studies with a number of NAD(H)- and ATP-dependent enzymes established that only the A and B rings
chemicals
commodity
contributed significantly to protein binding’“.“. Despite these uncertainties, the dye is a very sensitive spectroscopic probe tar the binding sites of enzymes. Designer dyes To date, ahnost all studies have been performed with commercia!!y acai!abLe dyes, designed originally for the texSie and printing industries. The selectivity of these afTmity ligands could, in principle, be improved by designing and synthesizing dyestufi which mimic the structure and binding of natural biological ligands more sympatheticallyj. As a finr step towards designing entirely novel ‘biomimetic’ dyes, the interaction between horse liver alcohol dehydrogenase (ADH) and anabgues of Cibacron Blue F3G-A was investigated in detail’“. Afhnity labellingr9 and X-ray crystailography~c’ established that the parent dye binds in the NAD’-binding
I
--
Figure 1 The structure of Cibacron Blue RG-A: ringA is the anthraqutnor!e ring; ring 6 is Ute diamimbenzene s&henate ring faiso know as the ‘bridging’ ring]; ring C is the triaziw ring; and ring 0 is the teiminal ring.
._
444 reviews Table 2. Apparent affinities of terminal-ring analoguesof anthraqukane dyes for horse liver alcohol dehydrogenase(ADH)
0.06 0.2
rn-coo-
H c-coo&So3mso; m-CH,OH mCONH, $0””
::: 1.6 s 5:9 9.3 lr1.5 172.2
pPO$P-NYCH,)~
site of the enzyme with the anthraquinone, diaminobenzrne sulphonate, and triazine rings (rings A, B and C, respectively, in Fig. I), apparently adopting similar positions as the adenine, adcnosine ribose and pyrophosphate groups of NAD’. The anthraquinone ring (A) binds in a wide apolar cleft which
Figure2 The putative binding pocket forthe terminalring analogue (mCOOS) of Cibacron Blue F3GAin the coenzyme binding site of horse liver alcohol dehydrogenase (ADH).The site lies lateral to the main coenzyme bin&g siie and comprise? the side chains of two juxtaposed cationicresidues Rrg47 and His51. TBTECH
CECEKBER
1992 WOL 10)
constitutes, -at_one end, the adcnine binding site, whilst the bridging ring (B) is positioned such that its sulphonate gn~~p intciacts with the guanidinium side chain of Arg271. Ring C binds close to where the pyrophosphate bridge ofthe coenzyme binds with the reactive triazinyl chlorine adjacent to the nicotinamide ribose binding site. The terminal ring (D) appears to be b&d in a cleft between the catalytic and coenzyme binding domains, with a possible interaction of the sulphonate with the side chain of Arg369. The binding of Cibacron Blue F3G-A to horse liver ADH resembles ADP binding, but differs significantly at the nicotinamide end of the molecule, with the mid-point position of ring D displaced from the mid-point position of the nicotinamide ring of NAD+ by about 1Ow’o. Consequently, a number of terminal-ring analogues of Cibacron Blue F3G-A were synthesized and characterized in an attempt to improve the specificity of dye binding tG the enzymeIs. Terminal-ring analogues bearing sulphonate, carboxylate, phosphonate, carboxamide and trimethyl-ammonium subsdtuents on the terminal ring were synthesized de IWVCfrom the key intermediate in the synthesis of anthraquinone dyestuf& bromamine acid, by sequensulphonatc, tial reaction with 1,Cdiaminobenzene cyanaric chloride and the appropriate arylaminesl*. In each case, the riye analagues were purified to >95% homogeneity and their affinity for horse liver ADH determined by difference spectroscopy at 25°C. Table 2 ids a number of terminal-ring analogues and their apparent dissociation constants which were deduced spcctrophotometrically. The data demonstrated that small substituents bind more tightly than bulkier species, especially if substibltrd in th-_ G- or itipositions with a neutral or anionic group. Ssbstitutions at the y-position produce analogues which display lower aff&ry for the enzyme; in particular, substitution with a cationic trimethylammonium group (Table 2, R = N*[CH,j,j produces a dye analogue with very low aftinity. The observed 2900fold variation in affinity of these anaiogues for the coenzymr binding site of horse liver ADH could be rationalized in terms of a putative binding pocket for the terminal ring comprising the side chains of two juxtaposed cationic residues, Arg47 and His51 (Fig. 2), and lying lateral to the main coenzyme binding site. This subsite encourages the binding of terminal rings bearing anionic sljbstituents, especially rhe M-COOgroup, in an otherwise hydrophobic pocket!x. inapection of the Structures shown in Fig. 2 and subsequent molecular modelling studies have shown that in the parent dye, Ci.bacron Blue FJG-A (Fig. 1) and the terminal-ring analogues, the ligands are too short and rigid to bind to horse liver ADH in a manner identical to the natural coenzyme, NAD’.. Consequently, analogues of the parent dye were designed and synthesized with central spacer functionalities to increase the length and flexibility of the molec&+.
These dyes
display higher affinity for h;irse liver ADH than the parent commercial dyes and mark a new horizon in ‘designer’ dyes for atSty chromatography.
Purification
of alkaline phosphatase
Immobilized commercial dyes have proven unsuitable for purifying alkaline phnsphatasr from calf intestinal mucosa: again neces&,;ng the introduction ofspecifically designed dyes as ligands”. A prelimjnary screen for the binding of crude enzyme’7 to immobilized commercial triazine dyes revealed an average 8.4-fold purification with enzyme r:coveries >90%. The average specific activity achieved (-25.2 pmol mg-1) fell far short of the specific activity of commercial ‘high purity’ preparations (850-l 25(!bmol mg’). Since carboxylate, phosphonav and boronatc arc known to be potent competitive inhibitors ofalkaline phosphatase, and the enzyme preferentially binds aromatic rather than aliphatic phosphate esters, substitution of these groups on dye chromophores would be expected to yield more effective adsorbents PrcIiminary evidence with immobilized terminal-ring analogues (Table 2; R = -Hg-COO-, m/p-SO,-, pCHPO,“, tn-EjOH],) showed that the paminobenzyl phosphonate analogue (Table 2, R = p-CH,PO,“-) proved superior for the purification of alkaline phospbatase, with up to a 200-fold increase in specific activity. Uy careful optimization ofadsorption and elution protocols, a 330-fold one-step purification of the enzyme from a crude calf intestinal extract was achieved using specific elution with inorganic phosphate (5 nM). The resulting ~lkalinr-phosphatasr preparahon displayed a sp~&fic activity in excess of 1000 pm01 mg-l , and was of equivalent purity to commercially available ‘high purity’ preparations and contained
reviews
Designerdyes:‘biomimetic’ s for the purificationof pharmaceutical proteinsby affinitychromatography Christopher R. Lowe, Steven J. Burton, Nicolas P. Burton, Wendy K. Alder-ton, Jennifer M. Pit-k and Janette A. Thomas_ Affinity chromatography has been extensively refined over the past few years to meet the more stringent criieria being placed on recombinant proteins as therapeutic products. New developments in the design of selective and stable ligands for affinity chromatography are establishing the technique as a routine tool in processscale protein purification. Exploitation of sophisticated molecular modelling kchniques in conjunction with binding and crystallographic studies has permitted the design of new, highly selective ‘bicmimetic’ ligands for the target proteins.
Few of the recent advances in biotechnology would have been commercially viable without &cient methods of protein purification. Substantia; worldwide markets exist for highly purified monoclonal antibodies, human growth factors, cytokines, hormones, blood clotting factors and fibtinolytics, erythropoietin, vacciiies and diagnostic en,zymes. The isolation and purification of such proteins;&e the most critical steps in he delivery of phannac&tical products with full biological activity and which meet the specifications for clinical applications. All steps in the production of pharmaceutical proteins must be in compliance with current Good Manufacturing Practices (GMP), regulated by the Food and Drug Administration (FDA) in the USA and by similar agencies elsewhere, and must ensure that the final product is i;llly active and unadulterated. Thus, biological molecules isolated from nattiral sources, or expressed from recombinant DNA systems, must be shown to have acceptably low levels of biologically active contaminants such as DNA ((3Opg per dose) and other macromolecules, viruses, pyrogen (~300 endotoxin units per dose) and ‘leachates’ Tom the separation media. Not surprisingl~~, therefore, the development of gentle, but effective, purification processes which yield fully active pharmaceutical proteins presents new challenges to the process engineer.
AfEnity chromatography In general, purification processes should be designed to produce the highest purity product at the highest attainable yield, since every percent loss of yield that occurs in the downstream processing is multiplied by all the manufacturing costs that came before’. Consequently, an eccnomic process may be contingent solely on thz effectiveness of the purification process that has been developed. Affinity techniques such as affinirr chromarography harness molecular recognition encountered between biological macromolecules and complementary ligands such as enzyme substrates, inhibitors, effecters, coenzymes, hormones, antigens, nucleic acids and sugars’J. tinity technology has been extensively developed over the past few years in response to greater demands being Imposed by the increased production of commercially valuable recombinant therapeutic proteins and the associated quality criteria. These more stringent requirements ofscale, yield and product purity have led to a critical reappraisal of many of the early ideas of affinity chromatographyl. Thus, the qualities of the support matrix, the activation and coupling chemistry, the selection and stability of the ligand and the purification format itself have all been re-examined in the light of these new requirements. In particular, the ligand selected for immobilizaticn has been scrutinized closely in recent yea&. A fimd;lmental advantage of affinity techniques is their predictive and rational character, since the ligandselected is designed to interact specificaliy with the protein to be purified. In principle, this should simplie process design and make the selective adsorption step much less sensitive to the
-ll~___l___ TIBTECH DECEMBER
1992 NO1 10)
0
1992. Elsewer Science Publishers Ltd (UK)
443 reviews Table 1. Comparisonof immobilizedIbiomimetic’dyes wilh conventionalaffinity adsorbents
composition of the fecdstork. However, in spite of over 20 years ofempirical development, the technique has only recently begun to find favour for the purification of high-value bioproducts. This is probably due, in part, to the inherent cost and lability of the ligands used, in part, to the troublesome problems of non-specific adsorption and fouling and, in part, to the difficulties associated with the sterilization and sanitization of relatively unstable adsorbents. Despite these limitations, affinity chromatography is making inroads into large-scale protein purification6~7. This review describes new developments in the design of selective and stable ligands for affinity chromatography which should alleviate some of the previous deficiencies of the technique and further establish it as a routine tool in process-scale protein purification.
Criterion
Immobilized ‘biomimatic’dyes
Conventional biospecilfic adsorbents
cost
Inexpensive,
Can be expensive
Stability
Chemidly and biologically stable
Tend to be labile
Specificity
Moderate
fvloderate to high
Synthesis of adsorbents
Facile, often onestep reaction
Often lengthy, synthetic route and/or toxic activation reagents
Capacity
High (>lQ% ligand utiiization)
Low (O.Ol-10% ligand utilization)
Tikmimetic ligtmds A&&y adsorbents created with naturally occurring
Scale-up potential
Virtually limitless
Very limited
biological hgands rend to be expensive to produce since the ligands themselves are chemically and biologically labile and are difhcult to immobilize in high yield with retention of activit@s. However, the exploitation of ‘pseudo- ’CT‘hiomimetic’ ligands in
Reusability
Very high
LOW
Sterilizability
High
Mostly low
place of their natural counterparts as ligands for affinity chromatography offers the prospect of circumventing many of these difficulties. The commonest class of ‘biomimetic’ &and is the reactive textile dyes which originate from the serendipitous discovery that the dye component of blue dextran, Cibacron Blue F3G-A, bound to pyruvate kinase during gel filtration9. This chance observation aroused interest in the dye, and subsequently !ed to the putiiCication of many proteins (including aibumin and other blood proteins, coenzyme-dependent enzymes, decarboxylases, glycolytic enzymes, hydrolases, lyases, nucleases, polymerases, synthetases and transferases)3Jt-33 on immobilized Cibacron Blue FSG-A affinity adsorbents. The use of immobiiized textile dyes offers significant advantages over conventionaI affinity adsorbents (Table 1). They are inexpensive, commodity chemicals, readily coupled to a variety of support matrices via their reactive groups and resistant to biological and chemica! degradation. Immobilized dye adsorbents display high capacities for their complementary protein and thus have considerable scale-up potential. The mechanism of binding of Cibacron Blue F3G-A to proteins is still unclear; originally, it was thought that the anthraquinone ring (ring A in Fig. 1) and the bridging diaminobenzene sulphonate ring (ringl? in Fig. 1) bore superficial similarity to the structure of ATPI*. It was later proposed that Cibacron Blue F3G-A might be a conformational analogue of the entire NAD’ molecule and thus might prove to be a diagnostic probe for the dinucleotide binding domain’s. However, this idea has been largely discredited since many proteins lacking this structural feature bind the dye, whilst some with the fold do not bind. Furthermore, kinetic and chromatographic studies with a number of NAD(H)- and ATP-dependent enzymes established that only the A and B rings
chemicals
commodity
contributed significantly to protein binding’“.“. Despite these uncertainties, the dye is a very sensitive spectroscopic probe tar the binding sites of enzymes. Designer dyes To date, ahnost all studies have been performed with commercia!!y acai!abLe dyes, designed originally for the texSie and printing industries. The selectivity of these afTmity ligands could, in principle, be improved by designing and synthesizing dyestufi which mimic the structure and binding of natural biological ligands more sympatheticallyj. As a finr step towards designing entirely novel ‘biomimetic’ dyes, the interaction between horse liver alcohol dehydrogenase (ADH) and anabgues of Cibacron Blue F3G-A was investigated in detail’“. Afhnity labellingr9 and X-ray crystailography~c’ established that the parent dye binds in the NAD’-binding
I
--
Figure 1 The structure of Cibacron Blue RG-A: ringA is the anthraqutnor!e ring; ring 6 is Ute diamimbenzene s&henate ring faiso know as the ‘bridging’ ring]; ring C is the triaziw ring; and ring 0 is the teiminal ring.
._
444 reviews Table 2. Apparent affinities of terminal-ring analoguesof anthraqukane dyes for horse liver alcohol dehydrogenase(ADH)
0.06 0.2
rn-coo-
H c-coo&So3mso; m-CH,OH mCONH, $0””
::: 1.6 s 5:9 9.3 lr1.5 172.2
pPO$P-NYCH,)~
site of the enzyme with the anthraquinone, diaminobenzrne sulphonate, and triazine rings (rings A, B and C, respectively, in Fig. I), apparently adopting similar positions as the adenine, adcnosine ribose and pyrophosphate groups of NAD’. The anthraquinone ring (A) binds in a wide apolar cleft which
Figure2 The putative binding pocket forthe terminalring analogue (mCOOS) of Cibacron Blue F3GAin the coenzyme binding site of horse liver alcohol dehydrogenase (ADH).The site lies lateral to the main coenzyme bin&g siie and comprise? the side chains of two juxtaposed cationicresidues Rrg47 and His51. TBTECH
CECEKBER
1992 WOL 10)
constitutes, -at_one end, the adcnine binding site, whilst the bridging ring (B) is positioned such that its sulphonate gn~~p intciacts with the guanidinium side chain of Arg271. Ring C binds close to where the pyrophosphate bridge ofthe coenzyme binds with the reactive triazinyl chlorine adjacent to the nicotinamide ribose binding site. The terminal ring (D) appears to be b&d in a cleft between the catalytic and coenzyme binding domains, with a possible interaction of the sulphonate with the side chain of Arg369. The binding of Cibacron Blue F3G-A to horse liver ADH resembles ADP binding, but differs significantly at the nicotinamide end of the molecule, with the mid-point position of ring D displaced from the mid-point position of the nicotinamide ring of NAD+ by about 1Ow’o. Consequently, a number of terminal-ring analogues of Cibacron Blue F3G-A were synthesized and characterized in an attempt to improve the specificity of dye binding tG the enzymeIs. Terminal-ring analogues bearing sulphonate, carboxylate, phosphonate, carboxamide and trimethyl-ammonium subsdtuents on the terminal ring were synthesized de IWVCfrom the key intermediate in the synthesis of anthraquinone dyestuf& bromamine acid, by sequensulphonatc, tial reaction with 1,Cdiaminobenzene cyanaric chloride and the appropriate arylaminesl*. In each case, the riye analagues were purified to >95% homogeneity and their affinity for horse liver ADH determined by difference spectroscopy at 25°C. Table 2 ids a number of terminal-ring analogues and their apparent dissociation constants which were deduced spcctrophotometrically. The data demonstrated that small substituents bind more tightly than bulkier species, especially if substibltrd in th-_ G- or itipositions with a neutral or anionic group. Ssbstitutions at the y-position produce analogues which display lower aff&ry for the enzyme; in particular, substitution with a cationic trimethylammonium group (Table 2, R = N*[CH,j,j produces a dye analogue with very low aftinity. The observed 2900fold variation in affinity of these anaiogues for the coenzymr binding site of horse liver ADH could be rationalized in terms of a putative binding pocket for the terminal ring comprising the side chains of two juxtaposed cationic residues, Arg47 and His51 (Fig. 2), and lying lateral to the main coenzyme binding site. This subsite encourages the binding of terminal rings bearing anionic sljbstituents, especially rhe M-COOgroup, in an otherwise hydrophobic pocket!x. inapection of the Structures shown in Fig. 2 and subsequent molecular modelling studies have shown that in the parent dye, Ci.bacron Blue FJG-A (Fig. 1) and the terminal-ring analogues, the ligands are too short and rigid to bind to horse liver ADH in a manner identical to the natural coenzyme, NAD’.. Consequently, analogues of the parent dye were designed and synthesized with central spacer functionalities to increase the length and flexibility of the molec&+.
These dyes
display higher affinity for h;irse liver ADH than the parent commercial dyes and mark a new horizon in ‘designer’ dyes for atSty chromatography.
Purification
of alkaline phosphatase
Immobilized commercial dyes have proven unsuitable for purifying alkaline phnsphatasr from calf intestinal mucosa: again neces&,;ng the introduction ofspecifically designed dyes as ligands”. A prelimjnary screen for the binding of crude enzyme’7 to immobilized commercial triazine dyes revealed an average 8.4-fold purification with enzyme r:coveries >90%. The average specific activity achieved (-25.2 pmol mg-1) fell far short of the specific activity of commercial ‘high purity’ preparations (850-l 25(!bmol mg’). Since carboxylate, phosphonav and boronatc arc known to be potent competitive inhibitors ofalkaline phosphatase, and the enzyme preferentially binds aromatic rather than aliphatic phosphate esters, substitution of these groups on dye chromophores would be expected to yield more effective adsorbents PrcIiminary evidence with immobilized terminal-ring analogues (Table 2; R = -Hg-COO-, m/p-SO,-, pCHPO,“, tn-EjOH],) showed that the paminobenzyl phosphonate analogue (Table 2, R = p-CH,PO,“-) proved superior for the purification of alkaline phospbatase, with up to a 200-fold increase in specific activity. Uy careful optimization ofadsorption and elution protocols, a 330-fold one-step purification of the enzyme from a crude calf intestinal extract was achieved using specific elution with inorganic phosphate (5 nM). The resulting ~lkalinr-phosphatasr preparahon displayed a sp~&fic activity in excess of 1000 pm01 mg-l , and was of equivalent purity to commercially available ‘high purity’ preparations and contained
