Báo cáo Y học: Galactosyl-mimodye ligands for Pseudomonas fluorescens b-galactose dehydrogenase pot

Galactosyl-mimodye ligands for Pseudomonas fluorescens b-galactose dehydrogenase Design, synthesis and evaluation C. F. Mazitsos 1 , D. J. Rigden 2 , P. G. Tsoungas 3 and Y. D. Clonis 1 1 Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, Greece; 2 Embrapa Recursos Gene ´ ticos e Biotecnologia, Brası ´ lia, Brazil; 3 Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, University of London, UK Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-bio- mimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appro- priate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomi- metic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic ÔlinkerÕ molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature ÔspacerÕ molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Fol- lowing their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsor- bents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescens b-galactose dehydrogenase. In addition, these mimodye affinity adsor- bents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-lig- ands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromato- graphic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purifi- cation of galactose dehydrogenase. Keywords: affinity chromatography; biomimetic ligands; galactose dehydrogenase; molecular modelling; triazine dyes. Galactose dehydrogenase (GaDH; D -galactose: NAD + 1-oxidoreductase; EC 1.1.1.48) catalyses the dehydrogena- tion of b- D -galactopyranose in the presence of NAD + to D -galacto-1,5-lactone and NADH, acting on the C1 posi- tion of the sugar substrate. The enzyme generally shows no absolute specificity either for NAD + ,asNADP + is also used, albeit to a lesser degree. Nor is the enzyme specific for D -galactose, as D -fucose is a better substrate, although other sugars (e.g. L -arabinose, 2-deoxy- D -galactose) are less reactive. The kinetic mechanism is ordered Bi-Bi, with the NAD + binding first to the enzyme [1]. GaDH from Pseudomonas fluorescens is the best studied example, as it has been cloned and expressed in Escherichia coli [2] and its full nucleotide sequence determined [3]. The active macro- molecule possesses two binding sites [4] and consists of two identical subunits each of 33 kDa (304 amino-acid residues) [3]. GaDH from Pseudomonas saccharophila has been studied to a lesser extent [5], whereas the enzyme has been identified in plants (e.g. green peas, oranges and Arabidopsis thaliana), algae (e.g. Iridophycus flaccidum) and several mammals including humans. No information is available regarding the catalytic mechanism of GaDH, and its structure has not been determined experimentally or modelled. GaDH is an important analytical tool as at alkaline pH the product galactonolactone is hydrolysed, so that the reaction becomes irreversible. The enzyme is therefore useful for the determination of b- D -galactose and a- D -galactose, after the latter is converted to the former by the application of exogenous mutarotase. GaDH is also exploited for the determination of lactose; the milk sugar is hydrolysed by lactase, coupled to GaDH which acts on the resulting b- D -galactose. Despite the utility of GaDH, a simple and rapid purification method is not available. The ability to combine knowledge of X-ray crystallo- graphic studies, NMR and homology structures with defined or combinatorial chemical synthesis and advanced computational tools has made rational design of affinity ligands more feasible, powerful, logical and faster [6]. In the present work, rigorous protein molecular modelling was Correspondence to Y. D. Clonis, Laboratory of Enzyme Technology, Department of Agricultural Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, Greece. Fax: + 30 210 5294307, Tel.: + 30 210 5294311, E-mail: clonis@aua.gr Abbreviations: ADH, alcohol dehydrogenase; BM, biomimetic ligand or mimodye ligand; CB3GA, Cibacron blue 3GA; GaDH, galactose dehydrogenase; GaO, galactose oxidase; GFO, glucose-fructose oxidoreductase; GlDH, glucose dehydrogenase; GlO, glucose oxidase; VBAR, Vilmafix Blue A-R; CDI, 1,1¢-carbonyldiimidazole. Enzymes: galactose dehydrogenase (GaDH; D -galactose: NAD + 1-oxidoreductase; EC 1.1.1.48). (Received 31 May 2002, revised 16 August 2002, accepted 28 August 2002) Eur. J. Biochem. 269, 5391–5405 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03211.x used to create an objectively sound model of GaDH using as the best available template glucose-fructose oxidoreductase (GFO). This model was then exploited in the design of novel galactosyl-biomimetic chlorotriazine dye-ligands (mimodye ligands) with bifunctional or chimaeric characteristics. In particular, these galactosyl-mimodye ligands are designed to bear a structural portion that interacts with the NAD + - binding site and a biomimetic moiety that interacts with the sugar-binding site of GaDH. The effectiveness of the bifunctional (chimaeric) ligand concept has been previously demonstrated with ketocarboxyl- [7,9] and glutathionyl- biomimetic [10] ligands but never with sugar ones. These mimodye ligands are expected to become useful tools for the identification of amino-acid residues of the binding sites of GaDH after affinity labelling. For this purpose, the galactosyl-mimodyes were designed to bear a reactive chloro-triazine structural scaffold, present in all reactive triazinyl-dye ligands including the archetypal CB3GA and VBAR. Other mimodyes and certain conventional triazine dyes are known to act as affinity labels due to their chlorine(s) atom(s) which react with appropriate residues of the targeted enzyme active site [11–13]. Furthermore, when the chlorine was substituted with a carefully chosen spacer molecule, a nonreactive biomimetic ligand was obtained which could be immobilized on a chromatography support. We envisage that these immobilized ligands will be of great use in the purification of GaDH from different sources. EXPERIMENTAL PROCEDURES Materials b-Galactose dehydrogenase (EC 1.1.1.48, P. fluorescens gene expressed in E. coli), galactose oxidase crude lyophi- lized powder (EC 1.1.3.9, from Dactylium dendroides), glucose oxidase crude lyophilized powder (EC 1.1.3.4, from Aspergillus niger, crude), D (+)-galactosamine (2-amino-2-deoxy- D -galactopyranose; chondrosamine), D (+)-galactose (minimum 99%), D (+)-glucose, 1,3-diamino- 2-hydroxypropane, bromoacetic acid N-hydroxysuc- cinimide ester, e-amino-n-caproic acid, ethylene-diamine, 1,5-diaminopentane, 1,6-hexane-diamine, 1,12-diaminodo- decane, 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC), 1,1¢-carbonyldiimidazole, o-tolidine, o-dianisi- dine, lipophilic Sephadex LH-20, CM–Sepharose CL-6B and DEAE–Sepharose CL-6B were obtained from Sigma (St Louis, MO, USA). All other diaminoalkanes were obtained from Aldrich (USA), whereas, shikimic acid was obtained from Fluka (USA). Peroxidase (from horseradish, grade I), NAD + (crystallized lithium salt c. 100%) and crystalline bovine serum albumin (fraction V) were obtained from Boehringer Mannheim (Germany). Hexylamine and nutrient broth (for microbiology) were obtained from Merck (Germany). The agarose chromatography gel Sepharose CL-6B was obtained from Pharmacia. F324 P. fluorescens biovar V1 was kindly donated by G. J. Nychas (Laboratory of Microbiology and Biotechnology of Foods, Agricultural University of Athens). Baker’s yeast, green peas and rabbit liver were purchased at the local market. Glucose dehydrogenase was extracted from P. fluorescens and baker’s yeast, while alcohol dehydro- genase was extracted from baker’s yeast and green peas. Protein modelling Fold recognition methods [14–17] were employed to deter- mine the best template to use for construction of a model of GaDH. Given the low sequence identity between GaDH and the GFO template used (17%) a rigorous modelling strategy was used, as previously (e.g [18,19]). In this way the challenge of modelling based on low sequence identity was met with a strategy designed to maximize model accuracy. Although errors will undoubtedly remain, the probability of producing a useful model is thereby enhanced. The essential elements of this strategy are the construction and analysis of multiple models (20 in this case), derived from limited randomization of initial coordinates and made with the program MODELLER [20], followed by analysis of packing and solvent exposure characteristics with PROSA II [21]. The resulting profiles showed regions of unusual protein struc- ture characteristics as peaks attaining positive values. These regions may result from locally inaccurate target-template alignment so that variant alignments, altered in these doubtful regions, were tested through further cycles of model construction and analysis. When better PROSA results were obtained for the variant alignment it was assumed to be more correct than the original. Stereochemical analysis using PROCHECK was also employed, particularly when the optimal target-template alignment had been reached. Pro- tein models were visualized using O [22]. Structurally similar proteins to the template were sought in the FSSP database (http://www.ebi.ac.uk/dali/fssp) [23]. STRIDE [24] was used for the definition of secondary structure. Ligand design and docking The ideal biomimetic would combine moieties that bind both to the cofactor NAD and the substrate binding sites. The initially considered Ôbuilding elementsÕ were two com- mercially available compounds: (a) anthrquinone-diamino- benzosulfonyl-dichlorotriazine (Vilmafix blue A-R or VBAR) containing three of the four ring systems of the well known dye Cibacron Blue 3GA (CB3GA), both known binding mimics of NAD(P) [8,9,25] and (b) 2-amino- 2-deoxygalactose, a substrate of GaDH [1]. Both these molecules have readily modifiable chemical groups to which could be attached an appropriate ÔlinkerÕ molecule in order to effect their fusion. 1-Amino-1-deoxygalactose, although commercially available, was not considered as GaDH attacks at the C1 position of the substrate, so that this position was thought better preserved in the ligand. However, in place of galactose shikimic acid was considered which, although only moderately structurally similar to GaDH substrates, has a clear advantage over them in terms of chemical stability. Finally, a ÔspacerÕ molecule of appro- priate length and chemical nature was designed to chemi- cally attach the complete ligand, via its triazine group (ring 3), to the chromatographic matrix. The HIC-UP database of heterocompounds [26] was used as a source of the Cibacron Blue-derived, b- D -galactose and shikimic acid components. These were rotated and translated with respect to the protein model using O [22] until optimal steric and chemical complementarity was reached. The tendency of Cibacron Blue-like ring systems to bind in NAD(P) binding sites with anthraquinone mimicking adenine, along with biochemical data regarding 5392 C. F. Mazitsos et al. (Eur. J. Biochem. 269) Ó FEBS 2002 sugar binding to related enzymes provided useful informa- tion to guide the docking, as described later. Side chain reorientations to rotameric conformations were allowed where they significantly enhanced interactions with ligands. The mimodye ligands (e.g. BM1 and BM2) were mod- elled through the fusion of their respective enzyme-bound components and the resulting complexes refined using CNS [27]. Topology and parameter files for energy minimization of the ligand were generated using XPLO 2 D [28] and hand- edited to reflect ideal stereochemical values. Synthesis and purification of the dye-ligands Amino-alkyl-VBAR dyes. (Table 1, structures aVBAR- fVBAR). Solid commercial VBAR (50 mg, 0.045 mmol dichloroform, purity 61.3%, w/w) was added to cold water (2 mL) and the solution was slowly introduced under stirring to a solution (3 mL) of the alkyl-diamines (0.73 mmol). The pH was adjusted to 8.9–9.0 and kept at this value with NaOH (0.1 M ) until the end of the reaction (2.5–3 h, 25 °C). The progress of each reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- wate, 2 : 4 : 1 : 3 v/v/v/v) upon completion of the reaction, solid NaCl was added (final content 3%, w/v) and the mixture was left at 4 °C. The pH of the mixture was adjusted with HCl (1 M ) to 1.0 and the precipitate was filtered (Whatman paper filter 50, hardened), washed with 5mLeachofHCl(1 M ) and cold acetone, then with 7 mL of diethyl ether and dried under reduced pressure. The solid dye (approximately 30 mg) was dissolved in 50 : 50 water/ methanol (50%) and dimethylsulfoxide (50%) mixture, and purified on a lipophilic Sephadex LH-20 column (30 · 2.5 cm) [29]. The purified product was stored in a desicator at 4 °C. Hydrophilic spacer-VBAR dye. (Table 1, structure gVBAR; Fig. 1). Stage 1: solid commercial VBAR (20 mg, 0.018 mmol dichloroform, purity 61.3%, w/w) was added to cold water (1 mL) and the solution was slowly introduced under stirring to 1,3-diamino-2-hydroxypropane (3 mL, 0.29 mmol). The pH was adjusted to 8.9–9.0 and kept at this value with NaOH (0.1 M ) until the end of the reaction (2.5–3 h, 25 °C). The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the dye was purified according to the method already described (see above). Stage 2: the purified product, 1,3-diamino-2-hydroxypropano-VBAR, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v) and the pH was adjusted to 7.5 with NaOH (0.1 M ). 0.2 mmol of bromoacetic acid N-hydroxysuccinimide ester [30,31] were dissolved in dioxane (1 mL) and this solution was Table 1. The structures of amino-alkyl-VBAR dyes (a-fVBAR), hydrophilic spacer-VBAR dye (gVBAR), galactosamine-VBAR dye and archetypal VBAR dye. Ligand R 1 R 2 a aVBAR –NH-(CH 2 ) 2 -NH 2 –NH 2 bVBAR –NH-(CH 2 ) 4 -NH 2 –NH 2 cVBAR –NH-(CH 2 ) 6 -NH 2 –NH 2 dVBAR –NH-(CH 2 ) 8 -NH 2 –NH 2 eVBAR –NH-(CH 2 ) 10 -NH 2 –NH 2 fVBAR –NH-(CH 2 ) 12 -NH 2 –NH 2 gVBAR –NH 2 Galactosamine-VBAR b –Cl VBAR –Cl (– NH 2 ) a –Cl a Following ligand immobilization, the -NH 2 group has replaced the -Cl atom. b The galactosamine-VBAR dye was synthesized employing the procedure for amino-alkyl-VBAR dyes but using the amino-sugar instead the diamino-alkane. Ó FEBS 2002 Galactosyl-mimodyes for galactose dehydrogenase (Eur. J. Biochem. 269) 5393 introduced to the dye solution. The pH was maintained to 7.5 until the end of the reaction (1.5 h, 4 °C, as judged by TLC). The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/ v/v). Upon completion of the reaction, the mixture was lyophilized and the dye was purified on the lipophilic Sephadex LH-20 column [29]. Stage 3: the purified product, bromoacetylated 1,3-diamino-2-hydroxypropano-VBAR, was dissolved in 0.1 M NaHCO 3 , pH 9.0 (2 mL) and the solution was slowly introduced under stirring to a solution of 0.4 M 1,3-diamino-2-hydroxypropane in 0.1 M NaHCO 3 , pH 9.0 (2 mL), while maintaining the pH to 9.0 with HCl (1 M ). The solution was then left under stirring for another 48–72 h (25 °C), without further adjustment of the pH. The progress of the reaction was monitored by TLC (1-butanol- 2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the dye was purified according to the method already described (see above). Biomimetic dye BM1. (Table 2, structure BM1; Fig. 1) Stage 1: purified hydrophilic spacer-VBAR, structure g (approx. 15 mg, 0.017 mmol) was dissolved in dimethyl- sulfoxide/water (3 mL, 50 : 50, v/v) and the solution was introduced under stirring to e-amino-n-caproic acid (2 mL, 0.17 mmol). The pH was adjusted to 9.0 and the mixture was left shaking at 60 °C for 3 h. The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the dye was purified according to the method already described (see above). Control dye C 6 gVBAR (Table 2) was synthesized in the same way. Stage 2: the purified product obtained from stage 1, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v), introduced to a solution of D (+)-galactosamine (3 mL, 0.62 mmol), and the pH was adjusted to 4.6, before freshly prepared solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.3 mL, 250 mg) was introduced dropwise under stirring over a period of 5 min, while maintaining the pH at 4.6–5.0. The reaction was stirred for 20 h at 25 °C without pH adjustment and monitored by TLC (1-butanol-2-propanol- ethylacetate-water, 2 : 4 : 1 : 3 v/v/v/v). A silver nitrate ammonia solution was used as a spray reagent for detecting the galactose-analogue in the newly synthesized dye [32]. The product, structure BM1, was precipitated by addition of solid NaCl (final content 15%, w/v), filtered and washed with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold acetone, and dried under reduced pressure. The product was re-suspended in 2 mL of water and precipitated by addition of solid NaCl (final content 10%, w/v). The precipitate was filtered and washed with 7 mL each of NaCl solution (10%, w/v) and cold acetone, desiccated with 7 mL of diethyl ether and dried under reduced pressure. Biomimetic dye BM2. (Table 2, structure BM2; Fig. 2). Stage 1: solid commercial VBAR (20 mg, 0.018 mmol dichloroform, purity 61.3%, w/w) was added to cold water (1 mL) and the solution was slowly introduced under stirring to a solution (3 mL) of 1,3-diaminopropane (0.29 mmol). The pH was adjusted to 8.9–9.0 and kept at this value with NaOH (0.1 M ) until the end of the reaction (2.5–3 h, 25 °C). The progress of each reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the dye was purified according to the method already described (see above). Stage 2: the purified product, VBAR-1,3-diaminopropane, was dissolved in dimethylsulf- oxide/water (3 mL, 50 : 50, v/v), introduced to a solution of shikimic acid (3 mL, 0.62 mmol), and the pH was adjusted to 4.6, before freshly prepared solution of 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (0.3 mL, 250 mg) was introduced dropwise under stirring over a period of 5 min, while maintaining the pH at 4.6–5.0. The reaction was stirred for a further 20 h at 25 °C without pH adjustment and monitored by TLC (1-butanol-2-propanol-ethylacetate- Fig. 1. Steps for the synthesis of gVBAR dye and of mimodye BM1. 5394 C. F. Mazitsos et al. (Eur. J. Biochem. 269) Ó FEBS 2002 water, 2 : 4 : 1 : 3 v/v/v/v). The product, VBAR-1,3-diami- nopropano-shikimic acid, was precipitated by addition of solid NaCl (final content 15%, w/v), filtered and washed with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold acetone, and dried under reduced pressure. The product was dissolved in a 50 : 50 water:methanol (50%) and dimeth- ylsulfoxide (50%) mixture, and purified to homogeneity on a lipophilic Sephadex LH-20 column (30 · 2.5 cm) [29]. Control dyes C 6 NgVBAR and C 3 NgVBAR (Table 2) were synthesizedinthesamewasasinstages1and2.Stages3–5: the purified product, VBAR-1,3-diaminopropano-shikimic acid, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v/v) and the solution was introduced under stirring to 1,3-diamino-2-hydroxypropane (2 mL, 0.17 mmol). The pH was adjusted to 9.0 and the mixture was left shaking at 60 °C for 3 h. The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the dye was purified according to the method already described (as above). The purified product, 1,3- diamino-2-hydroxypropano-VBAR-1,3-diaminopropano- shikimic acid, was dissolved in dimethylsulfoxide/water (3 mL, 50 : 50, v/v) and the pH was adjusted to 7.5 with NaOH (0.1 M ). 0.2 mmol of bromoacetic acid N-hydroxy- succinimide ester were dissolved in dioxane (1 mL) and this solution was introduced to the dye solution. The pH was maintained at 7.5 until the end of the reaction (1.5 h, 4 °C, as judged by TLC). The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate- water, 2 : 4 : 1 : 3 v/v/v/v). Upon completion of the reaction, the mixture was lyophilized and the dye was purified by applying preparative TLC as follows: lyophilized reaction mixture was dissolved in dimethylsulfoxide/water (0.4 mL, 50 : 50, v/v) and the solution applied on a Kieselgel 60 plate (silica gel 60, 0.2 mm, 20 · 20 cm, Merck). The plate was developed using a 1-butanol-2- propanol-ethylacetate-water (2 : 4 : 1 : 3 v/v/v/v) mixture. Following completion of the chromatography, the plate was dried and the band of interest was scraped off. The desired dye was extracted from the silica gel with water, filtered through a Millipore cellulose membrane filter (0.45 lm pore size) and lyophilized. The purified product, bromoacetylated 1,3-diamino-2-hydroxypropano-VBAR-1,3-diaminopropano- shikimic acid, was dissolved in 2 mL of 0.1 M NaHCO 3 , pH 9.0, and the solution was slowly introduced under stirring to a 2-mL solution of 0.4 M 1,3-diamino-2-hydroxy- propane in 0.1 M NaHCO 3 , pH 9.0 (the pH maintained at 9 using 1 M HCl). The solution was then left under stirring for another 48–72 h (25 °C), without further adjustment of the pH. The progress of the reaction was monitored by TLC (1-butanol-2-propanol-ethylacetate-water, 2 : 4 : 1 : 3 v/v/ v/v). Upon completion of the reaction, the product, hydrophilic spacer-VBAR-1,3-diaminopropano-shikimic acid, was precipitated by addition of solid NaCl (final content 15%, w/v), filtered and washed with 7 mL of NaCl solution (15%, w/v) and 5 mL of cold acetone, and dried under reduced pressure. The product was re-suspended in 2 mL of water and precipitated by addition of solid NaCl (final content 10%, w/v). The precipitate was filtered and washed with 7 mL each of NaCl solution (10%, w/v) and cold acetone, desiccated with 7 mL of diethyl ether and dried under reduced pressure. Table 2. The structures of the mimodyes BM1 and BM2 and the control dyes. Dye-ligand –R BM1 BM2 C 6 gVBAR C 6 NgVBAR C 3 NgVBAR Ó FEBS 2002 Galactosyl-mimodyes for galactose dehydrogenase (Eur. J. Biochem. 269) 5395 Spectroscopic characterization and analysis of dye-ligands Prior to their characterization, all dyes synthesized in this work were purified by following appropriate purification procedures, depending on the requirements of each syn- thetic step. During the preliminary purification stage, inorganic and certain organic contaminants were removed by extraction with ethyl ether and precipitation with acetone. In the next stage, complete dye purification was achieved on a Sephadex LH-20 lipophilic column, where salts and other organic impurities were removed. Prepara- tive TLC has also been used as a purification technique at certain stages. Successful dye purification was shown by TLC analysis (single blue bands). Tables 1–3 summarize the structures, molecular masses, molar absorption coefficients (e), and absorption maxima (k max ) of the purified free dyes. The absorption maxima (k max ) of the purified dyes were determined by aqueous dye aliquots (50 l M )takeninthe range 850–450 nm. The molar absorption coefficients (e-values) were calculated from the linear section of reference curves derived by plotting dye concentration vs. absorption (620 nm, 20–100 l M )[29]. NMR spectra. These were recorded on a BRUKER AM 250 or 500 MHz spectrometer using standard pulse sequences. Samples were analysed as solutions in dimethyl- sulfoxide-d 6 or D 2 O. The ABCD and ABX patterns of the aromatics of anthraquinone and 1,4-diamino-substituted phenyl rings, respectively, are expectedly present in the 1 H NMR spectra of all the compounds. The pattern is securely based by comparison with the 1 Hand 13 CNMR spectra of the commercially purchased reference com- pound VBAR. The 1 H NMR spectra of BM2 and BM1 show very complex, yet discernible high-field multiplets, ranging from d 1.05–4.85 p.p.m. and d 1.0–3.50 p.p.m., attributed to -CH-CH, -CH-NH and -CH-OH couplings, respectively. Multiplets at d 5.35–6.86 p.p.m. and d 5.20–6.80 p.p.m. are attributed to the amide –NH resonance of both BM2 and BM1, respectively. Mass spectra. Electron impact (EI) and fast atom bom- bardment (FAB) mass spectra were recorded on a VG ZAB/SE double focusing low/high resolution spectrometer. Electrospray ionization (ESI) spectra were run on a Finnigan LCQ DUO spectrometer. It is known that the reference compound VBAR does not exhibit a molecular ion (M + ) peak under EI ionization [49]. Indeed, no such ion has been observed in either EI, FAB or ESI spectra. Compounds BM2 and BM1 behaved similarly. However, comparing the highly complexed fragmentation patterns of BM2 and BM1, under the above ionization modes, allowed for the detection of fragments, resulting, most probably, from primary C–N and C–O fission. Immobilization of amino-dyes to carbonyldiimidazole- activated agarose and determination of immobilized dye concentration Agarose beads (Sepharose CL-6B) were activated with 1,1¢- carbonyldiimidazole (CDI) by a modification of the pub- lished method [33]. An immobilized dye concentration of approximately 2.0 lmolÆg )1 Sepharose CL-6B (moist gel) was achieved by using appropriate amount of CDI in the activation step. Exhaustively washed (300 mL of water) Sepharose CL-6B (600 mg, moist weight) was washed sequentially with dioxane-water (10 mL, 3 : 7, v/v), diox- ane-water (10 mL, 7 : 3, v/v), dioxane (10 mL) and dried dioxane (25 mL). The gel was re-suspended in dried dioxane (1.1 mL) to which CDI (200 mg) had already been added Fig. 2. Steps for the synthesis of mimodye BM2. 5396 C. F. Mazitsos et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and the mixture was tumbled for 1–2 h at 20–25 °C. Activated gel was washed with dried dioxane (20 mL) and used immediately. A solution of amino-dye (0.02 mmol) in dimethylsulfoxide/water (2 mL, 50 : 50, v/v) was adjusted to pH 10.0 with 2 M sodium carbonate solution, whereupon CDI activated Sepharose CL-6B (600 mg) was added. The mixture was tumbled overnight (25 °C) and washed sequentially with water (50 mL), NaCl solution (25 mL, 1 M ), water (25 mL), dimethylsulfoxide solution (6 mL, 50 : 50, v/v) and, finally, water (50 mL). In the case of ligands with remaining active Cl, the adsorbent, after the immobilization procedure, was suspended in NH 3 solution (1 M , pH 8.5) and tumbled for another 3 h. The dyed gels were stored as moist gels in 20% methanol at 4 °C. Table 3 summarizes the conditions and performance of immobil- ization reactions of the dye-ligands. Determination of immobilized dye concentration was achieved according to [34]. The concentration of the immobilized dyes was calculated as micromoles of dye per gram moist mass gel, using the molar absorption coefficients shown in Table 3. All adsorbents were substituted with dye-ligand at approximately the same level (1.8–2.3 lmol dyeÆgmoistgel )1 ). When comparing affinity adsorbents, equal ligand substitution effected by synthesis rather than dilution with unsubstituted gel is an important but often overlooked prerequisite. Wide var- iations in immobilized ligand concentration are undesir- able because the results obtained from the employment of such affinity adsorbents may lead to misleading conclusions. Extreme levels of ligand substitution may lead to no binding, due to the steric effect caused by the large number of dye molecules, or even to nonspecific protein binding [47,48]. Assay of enzyme activities and protein, and inactivation of galactose dehydrogenase by VBAR Galactose dehydrogenase (GaDH), galactose oxidase (GaO), glucose oxidase (GlO), glucose dehydrogenase (GlDH) and alcohol dehydrogenase (ADH) assays were performed at 25 °C with the exception of GlO, which was performed at 35 °C. The assays were performed according to [35] [36], [37], [38], and [39], respectively. All assays were performed in a double beam UV-visible spectrophotometer equipped with a thermostated cell holder (10-mm path- length). For GaO, one unit of enzyme activity is defined as the amount that produces a DA 425nm of 1.0 per min at the conditions of the assay. For the rest of the enzymes, one unit of enzyme activity is defined as the amount that catalyses the conversion of 1 lmol of substrate to product per min. Protein concentration was determined by the method of Bradford [40] or by a modified Bradford’s method [41], using bovine serum albumin (fraction V) as standard. Inactivation of GaDH by VBAR was performed in incubation mixture containing in 1 mL total volume (35 °C): 100 lmol Hepes/NaOH buffer pH 8.5, 30 nmol VBAR, 0.13 U GaDH (enzyme assay at 25 °C). The rate of GaDH inactivation was followed by periodically removing samples (100 lL) from the incubation mixture for assay of enzymatic activity. Competitive inactivation studies of GaDH by VBAR were performed in the above reaction mixture of 1 mL total volume (35 °C) containing also 1 lmol NAD + . Preparation of cell extracts with enzyme activities P. fluorescens dry cells (1.5 mg) were suspended in 1 mL of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 6.5, 7.0 or 7.5, and ultrasonically disintegrated (Vibra Cell, 400 Watt, Sonics & Materials) (amplitude: 40%, 2 s sonication, 5 s pause, 8 cycles, 4 °C).Celldebris was removed by centrifugation (5000 g,20min,4°C) and the supernatant was dialyzed overnight at 4 °Cagainst2L of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 6.5, 7.0 or 7.5. The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, 0.08 U GlDHÆmL )1 extract (0.05 U GlDHÆmg dry cells )1 ).InthecaseofGaDH, before dialysis, the supernatant was enriched as necessary with commercial enzyme (P. fluorescens gene expressed in Table 3. Characteristics of free biomimetic and nonbiomimetic dyes, and conditions and performance of their immobilization reactions onto agarose. Dye-ligand M r (sodium salt) me (m M )1 Æcm )1 ) in water k max (nm) in water mg dye per g moist gel (in reaction) lmol dye per g moist gel (in adsorbent) me a (m M )1 Æcm )1 ) BM1 1119 7.1 628 37.3 1.9 5.3 BM2 1057 6.8 616 35.2 1.8 4.9 C 6 gVBAR 928 5.9 622 30.9 2.1 4.2 C 6 NgVBAR 985 5.8 623 31.4 2.0 4.8 C 3 NgVBAR 943 6.0 618 28.1 2.2 6.3 aVBAR 685 5.5 619 22.8 2.2 4.4 bVBAR 713 5.7 621 23.8 2.3 4.3 cVBAR 741 5.4 622 24.7 2.1 4.5 dVBAR 769 5.8 622 25.6 1.8 4.1 eVBAR 797 5.6 623 26.6 2.0 3.9 fVBAR 825 5.3 620 27.5 1.9 4.2 gVBAR 844 8.0 621 28.1 2.2 6.3 a Determined in medium identical to the one that resulted after acid hydrolysis of the adsorbent. Values were determined from 20 l M dye solutions made in the above medium. The duration of all reactions was 18 h. Ó FEBS 2002 Galactosyl-mimodyes for galactose dehydrogenase (Eur. J. Biochem. 269) 5397 E. coli) in order to achieve an initial specific activity of about 1.1 U GaDHÆmg )1 . Commercial lyophilized crude powder (10 mg) of Dacty- lium dendroides was suspended in 2 mL of 100 m M potas- sium phosphate buffer, pH 7.0 or 7.5 and the suspension was centrifuged (5000 g,20min,4°C). The supernatant was dialyzed overnight at 4 °C against 2 L of 100 m M potassium phosphate buffer, pH 7.0 or 7.5. The dialysate was clarified through a Millipore cellulose membrane filter (0.45 lm pore size), affording specific activity, typically, of 51.3 U GaOÆmg )1 (18.3 U GaOÆmL )1 extract, 3.7 U GaOÆmg cell lyophilized powder )1 ). Commercial lyophilized crude powder (11 mg) of Aspergillus niger was suspended in 1.5 mL of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0 or 7.5. The suspension was centrifuged (5000 g, 20 min, 4C) and the supernatant was dialyzed over- night at 4 °C against 2 L of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0 or 7.5. The dialysate was clarified through a Millipore cellulose membrane filter (0.45 lm pore size), affording, typically, 11.2 U GlOÆmL extract )1 (1.5 U GlOÆmg solid )1 ). Commercial baker’s yeast cells (9 g paste) were suspen- ded in 12mL of 10m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0 or 7.5, or 10 m M potas- sium phosphate buffer, pH 6.5, 7.0 or 7.5, before ultra- sonically disintegrated (amplitude 40%, 5 s sonication, 5 s pause, 12 cycles, 4 °C).Celldebriswasremovedby centrifugation (14 000 g,50min,4°C) and the supernatant was dialyzed overnight at 4 °Cagainst2Lof10m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0 or 7.5, or 10 m M potassium phosphate buffer, pH 6.5, 7.0 or 7.5. The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, an activity of 0.06 U GaDHÆmL extract )1 (0.08 U GaDHÆgcellpaste )1 ), 0.39 U GlDHÆmL extract )1 (0.52 U GlDHÆg cell paste )1 )and5.5U ADHÆmL extract )1 (7.3 U ADHÆg cell paste )1 ). Green peas (13 g) were suspended in 20 mL of 10 m M potassium phosphate buffer containing 1 m M EDTA, 7.0 or 7.5, or 10 m M potassium phosphate buffer, pH 6.5, 7.0 or 7.5, before pulped using pestle and mortar, and homogenized (Virtishear mechanical homogenizer, 10 000 r.p.m., 1 min, 4 °C). The homogenized suspension was filtered using cheese cloth and the filtrate was centrifuged (18 000 g,40min,4°C). The supernatant was dialyzed overnight at 4 °C against 5 L of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0 or 7.5, or 10 m M potassium phosphate buffer, pH 6.5, 7.0 or 7.5. The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, an activity of 0.02 U GaDHÆmL extract )1 (0.03 U GaDHÆg )1 )and0.3UADHÆmL ex- tract )1 (0.46 U ADHÆg )1 ). Rabbit liver (5 g) was suspended in 20 mL of 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 6.5 or 7.0, and homogenized (Virtishear mechanical homogenizer, 10 000 r.p.m., 3 min, 4 °C). The homogen- ized suspension was centrifuged (750 g for 15 min, 4 °C) and the supernatant was re-centrifuged (14 000 g,50min, 4 °C). The supernatant was dialyzed overnight at 4 °C against 5 L of 10 m M potassium phosphate buffer, pH 6.5 or 7.0. The dialysate was clarified through a Milipore cellulose membrane filter (0.45 lm pore size), affording, typically, an activity of 0.03 U GaDHÆmL extract )1 (0.12 U GaDHÆg )1 ). Affinity chromatography evaluation of the amino-alkyl- dyes, hydrophilic spacer-dye and control-dyes using GaDH from P. fluorescens extract All procedures were performed at 4 °C. Galactose dehy- drogenase binding was assessed using analytical columns, each packed with 0.5 mL of adsorbent bearing immobilized ligand (amino-alkyl-VBAR dyes, structures aVBAR- fVBAR and hydrophilic spacer-VBAR dye, structure gVBAR of Table 1, as well as control-dyes, structures C 6 gVBAR, C 6 NgVBAR and C 3 NgVBAR of Table 2) (1.8– 2.3 lmol dyeÆgmoistgel )1 ). Columns were equilibrated with 10 m M potassium phosphate buffer containing 1 m M EDTA, pH 7.0. Dialyzed P. fluorescens extract (0.5– 1.0 mL, 0.1–0.2 U GaDH, 0.09–0.17 mg protein) was applied to each analytical column. Non-adsorbed protein was washed off with equilibration buffer (2–3 mL). Bound GaDH activity was eluted with 2 mL equilibration buffer containing a mixture of 1 m M NAD + and 10 m M Na 2 SO 3 . Collected fractions (1 mL) were assayed for GaDH activity. Affinity chromatography evaluation of mimodye adsorbents using GaDH from P. fluorescens extract All procedures were performed at 4 °C. Galactose dehy- drogenase binding was assessed using analytical columns, each packed with 0.5 mL of mimodye adsorbent (1.8–2.2 lmol dyeÆgmoistgel )1 ). Columns were equili- brated with 10 m M potassium phosphate buffer at the pHs shown in Table 4, containing 1 m M EDTA. Dialyzed P. fluorescens enriched extract (0.5–1.0 mL, 0.10–0.38 U GaDH, 0.09–0.33 mg protein) was applied to each analyt- ical column. Non-adsorbed protein was washed off with equilibration buffer (2–4 mL). Bound GaDH was eluted, from immobilized BM1, by a mixture of 0.5 m M NAD + and 5 m M Na 2 SO 3 in the equilibration buffer (2–4 mL) or, from immobilized BM2, by 0.8 m M NAD + and 8 m M Na 2 SO 3 in the equilibration buffer (3–4 mL). Collected fractions (1 mL) were assayed for GaDH activity and protein [41]. The fractions with GaDH activity were pooled and the specific activity was determined. Table 4. Affinity chromatography evaluation of immobilized mimodyes and hydrophilic spacer-VBAR dye for binding GaDH activity from P. fluorescens crude extract. Dye-ligand pH SA (unitsÆmg )1 ) Purification (-fold) Recovery (%) BM1 6.5 29.9 27.2 66 7.0 48.2 41.9 100 7.5 30.3 27.5 35 BM2 6.5 15.1 13.7 68 7.0 37.7 32.8 76 7.5 41.2 37.5 98 8.0 28.8 25.3 28 gVBAR 7.0 15.4 13.4 33 5398 C. F. Mazitsos et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Affinity chromatography control experiments for the evaluation of the binding selectivity of mimodye adsorbents using enzymes other than GaDH On each of the mimodye adsorbents (0.5 mL), previously equilibrated with 10 m M potassium phosphate buffer (pH 7.0 for BM1 and 7.5 for BM2) containing 1 m M EDTA, were applied the enzyme units shown in Table 5, previously dialyzed in the same equilibration buffer (4 °C). After the column was washed with equilibration buffer, elution of bound proteins was effected with 1 M KCl in the same buffer. In the case of GaO, the equilibration buffer used was 100 m M potassium phosphate (pH 7.0 for BM1, pH 7.5 for BM2). RESULTS AND DISCUSSION Protein molecular modelling Fold recognition results were near-unanimous in highlight- ing the structure of Zymomonas mobilis glucose-fructose oxidoreductase (GFO; PDB code 1ofg;) as the best available template for GaDH model construction. For example, the 3D-PSSM method [16] gave GFO a score of 6 · 10 )6 with the next best hit scoring 95 · 10 )3 . Similarly, the FFAS method [17] gave GFO a score of 67 and the next best template just 14. In each case, these results are strongly significant for GFO and show it to be much more suitable as GaDH template than the next best structures. The only exception to the trend was GENTHREADER [15] which gave GFO and rat biliverdin reductase similar high probabilites of 0.94 and 0.95, respectively. In fact GFO, rat biliverdin reductase and many others of the better scoring hits of the fold recognition studies, all catalyse redox reactions and are structurally related, sharing a dinucleotide binding fold, in conjunction with a more variable domain responsible principally for substrate binding [42]. Based on the FFAS alignment an initial GaDH-GFO target-template alignment was constructed by examination of the GFO structure to determine the most likely positions at which the 10 insertions or deletions could be accommo- dated. In most cases these positions were between secondary structure elements but in others, the size of the insertion or deletion naturally led to alteration of neighbouring helices or strands (see Fig. 3). Although GaDH is a dimer, the regions of the alternate subunit corresponding to those that Table 5. Control experiments for the evaluation of the binding selectivity of immobilized BM1 and BM2 with enzymes other than GaDH. On each affinity adsorbent (0.5 mL), previously equilibrated with 10 m M potassium phosphate buffer containing 1 m M EDTA (pH 7.0 for BM1, pH 7.5 for BM2), were applied the enzyme units shown, previously dialyzed in the same equilibration buffer as above (4 °C). After the adsorbent was washed with equilibration buffer, elution of bound proteins was effected with 1 M KCl. For GaO, 100 m M potassium phosphate buffer (pH 7.0 for BM1, pH 7.5 for BM2) was used as the equilibration buffer. BM1 BM2 Enzyme Source Units applied Bound enzyme (%) Units applied Bound enzyme (%) GaO Dactylium dendroides 15.7 5.8 13.8 5.2 GlO Aspergillus niger 8.3 6.2 4.6 12.6 GlDH P. fluorescens 0.1 4.9 0.1 4.8 GlDH Baker’s yeast 0.7 0.7 0.8 0.5 ADH Baker’s yeast 3.8 3.3 3.9 2.6 ADH green peas 0.3 19.4 0.3 15.9 Fig. 3. ALSCRIPT [52] alignment of GaDH with template GFO. The secondary structure of GFO is shown above the alignment and residues shared between the two proteins are emboldened. Ó FEBS 2002 Galactosyl-mimodyes for galactose dehydrogenase (Eur. J. Biochem. 269) 5399 in GFO contribute to cofactor and substrate binding (the N-terminal stretch and the loop around GFO residue 317), are not present in GaDH (Fig. 3). Therefore, modelling of an individual monomer was undertaken. The first set of 20 models was, thus, constructed and analysed as outlined in Materials and methods. At regions of improbable protein packing and solvent exposure, as indicated by PROSA II, a series of alignment variants was constructed. These variants, typically, involved 1–3 residues shifts of single secondary structure elements, with the flanking loops accommodating correspondingly altering in length. These were analysed and the process repeated until no further alignment improve- ments could be found. In all 17 different alignments were tested. Special attention was then paid to stereochemical aspects of the model. Residues in disallowed or generously allowed areas of the Ramachandran plot were treated as possible errors and dealt with either by flipping of peptide bonds or ab initio regeneration with MODELLER .Attheend of this process the structure best combining low PROSA II score and good stereochemistry was taken as the final model. As previously observed, significant improvements in model quality resulted from this careful construction procedure. The first set of models had PROSA II scores in the range )7.8 to )8.7. For the final model this improved to )10.1. Comparison with the template also suggests a model of high objective quality. The somewhat longer GFO (351 residues vs. 303 in the final model) scores )11.5 by PROSA II analysis. The overall stereochemical quality of the model and GFO, as measured by the G-factor calculated by PROCHECK , is near-identical; )0.15 for the model, )0.16 for the crystal structure. The GaDH model places 90.5% of residues in most-favoured regions of the Ramachandran plot, suggestive of good structural quality and similar to the 91.5% value of the GFO template. As well as these overall indicators, it is worth remembering that the isolated regions of high sequence identity between target and template, around GaDH positions 10 and 85 (Fig. 3), are situated near the cofactor binding site. Hence, this part of the final structure, important for docking studies, should be parti- cularly well-modelled. Ligand design and docking With the good objective quality of the GaDH model established, docking experiments were initiated to indicate possible galactosyl-biomimetic ligands for GaDH. The three ring systems of the CB3GA-derived portion (num- bered 1–3: anthraquinone, diaminobenzosulfonic acid and triazine, respectively; see Fig. 4A and Table 1, VBAR) were first docked into the GaDH model, followed sequentially by the galactose portion, the ÔlinkerÕ molecule between ring 3 and the galactose, and finally the chain (ÔspacerÕ molecule) by which the ligand attaches to the chromatograpic matrix (e.g. agarose beads). Experimental evidence regarding residues involved in substrate and cofactor binding to GaDH is entirely lacking, and inference of possible important regions through their sequence conservation is rendered impossible by the lack of any known close GaDH homologues. Nevertheless, a variety of other indirect data could be used to guide the docking. The knowledge that ring systems 1–3 bind in NAD(P) binding sites, with the anthraquinone ring system 1 Fig. 4. Stereo MOLSCRIPT [53] diagrams showing interactions of unre- fined, docked components with the final GaDH model. (A) Ring systems 1–3 (B) galactose (presumed to bind similarly to 2-amino-2-deoxyga- lactose) and (C) shikimic acid. Hydrogen bonds are shown by dotted lines. 5400 C. F. Mazitsos et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... from the dehydrogenase family (glucose dehydrogenase and alcohol dehydrogenase) The binding observed between the enzymes tested and mimodyes BM1 and BM2 was generally satisfactory low (Table 5) From the six enzymes tested, only ADH from green peas showed some binding (19.4% for BM1 and 15.9% for BM2) followed by glucose oxidase from A niger (6.2% for BM1 and 12.6% for BM2), with all other enzymes showing... Consequently, the experimental approach adapted here for a practically meaningful evaluation of the computationally predicted mimodye ligands for GaDH was that of affinity chromatography, using adsorbents bearing immobilsed biomimetic and nonbiomimetic/control ligands and using GaDH from P fluorescens crude extracts The two new galactosyl-mimodye adsorbents, BM1 and BM2, at pH values 7.0 and 7.5, respectively,... S.C., Rigden, D.J & Clonis, Y. D (2001) New family of glutathionyl-biomimetic ligands for affinity chromatography of glutathione-recognising enzymes J Chromatogr A 917, 29–42 11 Clonis, Y. D & Lowe, C.R (1980) Triazine dyes, a new class of affinity labels for nucleotide-dependent enzymes Biochem J 191, 247–251 12 Clonis, Y. D., Goldfinch, M.J & Lowe, C.R (1981) The interaction of yeast hexokinase with Procion... the dye adsorbents In view of the practical applications envisaged for the new mimodye ligands, we made further comparisons for the final models with experimental data obtained using agarose-immobilized ligands The affinity of an immobilized ligand for the complementary protein is determined Ó FEBS 2002 Galactosyl-mimodyes for galactose dehydrogenase (Eur J Biochem 269) 5403 Fig 6 MOLSCRIPT [53] diagrams... the 2-hydroxyl group suitably positioned to hydrogen bond to Tyr260 in accord with the experimental data [1] The 3- and 4-hydroxyl groups of the galactose thus positioned were suitably placed to hydrogen bond to the side-chain oxygen (OD1) and nitrogen atoms of Asn167 and Ile115, respectively One hydrophobic face of the sugar interacted favourably with the side chain of Trp225 [46] Little information... determination Acta Crystallogr D54, 905–921 28 Kleywegt, G.J & Jones, T.A (1997) Model-building and refinement practice Methods Enzymol 277, 208–230 29 Labrou, N.E & Clonis, Y. D (1995) The interaction of Candida boidinii formate dehydrogenase with a new family of chimeric biomimetic dye -ligands Arch Biochem Biophys 316, 169–178 30 Cuatrecasas, P & Anfinsen, C.B (1971) Affinity chromatography Methods Enzymol 22, 345–378... matrix for affinity chromatography devoid of additional charged groups J Biol Chem 254, 2572–2574 34 Labrou, N.E., Karagouni, A & Clonis, Y. D (1995) Biomimeticdye affinity adsorbents for enzyme purification: application to the one-step purification of Candida boidinii formate dehydrogenase Biotechnol Bioeng 48, 278–288 35 Wallenfels, K & Kurz, G (1962) Uber die spezifitat der galaktosedehydrogenase aus Pseudomonas. .. analytisches hilfsmittel Biochem Z 335, 559–572 36 Bergmeyer, H.U (1988) Galactose oxidase from Polyporus circinatus In Methods of Enzymatic Analysis (Bergmeyer, H.U., Bergmeyer, J & Grabl, M., eds), Vol II, 3rd edn, pp 194–195 VCH, Germany 37 Bergmeyer, H.U., Gawehn, K & Grassl, M (1974) Methods of Enzymatic Analysis (Bergmeyer, H.U., eds), Vol I, 2nd edn, pp 457 Academic Press Inc, New York, NY 38... and (B) BM2 Hydrogen bonds are shown by dotted lines partly by the characteristics of the ligand per se and partly by the solid support and coupling chemistry Studies with ligands free in solution do not fairly reflect the chemical, geometrical and steric constrains imposed by the complex 3D solid support environment [6] Not surprisingly therefore earlier studies have appeared contradictory [29,34,50]... Interestingly, while the binding of each enzyme was approximately the same for both mimodyes, for glucose oxidase from A niger, mimodye BM2 was double effective compared to BM1 The aforementioned figures are near and below those obtained for the nonbiomimetic control adsorbents with GaDH It is not unreasonable to expect that the large nonmimetic portion of the mimodye ligands may allow for some interactions . Galactosyl-mimodye ligands for Pseudomonas fluorescens b-galactose dehydrogenase Design, synthesis and evaluation C. F. Mazitsos 1 ,. chromatography materials for the target enzyme, Pseudomonas fluorescens b-galactose dehydrogenase. In addition, these mimodye affinity adsor- bents displayed good