Synthesis and characterization of soluble dextran-adenosine phosphate complexes: Kinetic effects of coenzyme loading

Synthesis and characterization of soluble dextran-adenosine phosphate complexes: Kinetic effects of coenzyme loading J. Hubble,* R. Eisenthal,t A. Herbert,t I. Tarrt and R. England* * School o f Chemical Engineering and ~ Biochemistry Group, University o f Bath, Bath, U K

Soluble dextran-ATP complexes have been synthesized using a bifunctional oxirane as the coupling agent. The degree of coupling is time-dependent, allowing materials of varying coenzyme loadings to be produced very simply. Characterization studies have shown that at the maximum coenzyme loading obtained (34 molecules per complex) all coenzyme moieties were coenzymically active with hexokinase. The extent of coenzyme loading was shown to have a considerable influence on the values of K m and Vmax o f the complex as a substrate for hexokinase. Enzyme activity was also found with acetate kinase and myokinase, and coenzyme recycling (ATP, ADP) was demonstrated in an ultrafiltration reactor.

Keywords:Dextran-ATP complexes;coenzymeloading

Introduction Much work has been published on the immobilization of coenzymes to insoluble supports, mainly for the purposes of affinity chromatography. Less attention has been directed at the attachment of coenzymes to soluble macromolecular matrices. Such derivatized coenzymes, when enzymically active, have particular application in ultrafiltration membrane reactors, biochemical fuel cells, or biosensors, and may increase the utility of these systems by extending the range of enzymes available for use. There has been particular interest in the provision of nicotinamide adenine dinucleotide [NAD(H)] for a number of commercially interesting enzyme reactions. Early workers concentrated on the attachment of NAD to soluble dextrans activated with cyanogen bromide. More recently other polymers have been used to form soluble high molecular weight complexes for use in ultrafiltration membrane reactors, including polyethylenglycol and a number of acrylic polymers. 2 Many of the early preparations suffered from low enzymic activity and it rapidly became clear that complexes which showed affinity interaction with enzymes could

Address reprint requests to Dr. Hubble at the School of Chemical Engineering, Universityof Bath, Bath, UK BA2 7AY Received II July 1988; revised 16 December 1988

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not necessarily support their full catalytic action? However, further developments have led to improved NAD complexes such that commercial reactors based on coenzyme requiring systems are now feasible4 and operating. 5,6 Although chemically similar, coenzyme complexes based on adenosine phosphates have been the subject of much less research and at present there are no commercial enzyme processes which utilize them. In principle, similar coupling chemistry is available for preparing soluble adenosine phosphate complexes. However, in practice it cannot be assumed that these will lead to levels of activity similar to those observed with NAD(H). Previous studies have demonstrated the synthesis of soluble complexes of adenosine phosphates 7-12 and their use in ultrafiltration reactors, 9'j2-j4 but more work is needed to allow a simple high-yield coupling procedure with characterized kinetic behaviour. Sundberg and Porath 15developed a method, using bis-oxiranes, for attaching ligands to insoluble polymers and applied this to the coupling of proteins, peptides, and other amino group-containing compounds to Sepharose 4B. This method has been modified to allow the synthesis of soluble derivatives of adenosine phosphates bound to dextran. We have investigated conditions that allow one to control the number of cofactor molecules bound per macromolecule in a reproducible manner, and we now report the preparation and characterization of ATP-dextran and ADP-dextran and their

© 1990

Butterworth Publishers

Dextran-A TP complexes: J. Hubble et al. specificity and kinetic behavior with several ATPdependent kinases.

Materials 1,4-Butanediol diglycidyl ether (DGE), technical grade, was obtained from Aldrich Chemical Co. The mean oxirane content as determined by thiosulfate titration (see Methods) was 1.7 mole per mole DGE. Dextran T40 was purchased from Pharmacia; an average molecular weight of 40,000 was assumed and used in all calculations. Enzymes and coenzymes were purchased from Sigma Chemical Co., Poole, Dorset, UK. Enzyme sources were as follows: hexokinase (yeast), glucose-6-phosphate dehydrogenase (L. mesenteroides), pyruvate kinase and myokinase (rabbit muscle), lactate dehydrogenase (pig heart), acetate kinase (E. coli). All other reagents were of analar grade and were obtained from British Drug Houses, Poole, Dorset, UK.

Methods Activation o f dextran Dextran T40 (20 g) was dissolved in 80 ml of 0.25 M NaOH containing 2 mg ml- ~sodium borohydride. Then 16 ml 1,4-butanediol diglycidol either was added dropwise over 30 rain at room temperature with continuous stirring. The reaction was allowed to proceed at room temperature and samples were withdrawn at specific time intervals (see Results). The reaction was terminated in these samples by addition of sufficient 2 M HCI to lower the pH to 5.5, and the activated dextran precipitated by addition of five volumes of cold absolute ethanol. The resulting suspension was allowed to stand on ice for 3 h, the ethanol then decanted, and the precipitates freeze-dried.

Epoxide assay This was carried out at pH 7.0 using the thiosulfate method described by Axen et al., j6 which is based on the titration of hydroxide ion released by the reaction of thiosulfate with oxirane groups. The assay was carfled out in a pH-stat (Radiometer). Typically, 10-15 mg of activated dextran was dissolved in 1 ml distilled water and, after adjustment of the pH to 7.0, 1 ml of 100 mM sodium thiosulfate was added; the amount of HCI required to maintain the pH at 7.0 was used to calculate the number of free epoxide groups. The use of a pH-stat was essential for reproducible results, as the assay was found to be highly sensitive to pH fluctuations.

Synthesis o f dextran-ATP and dextran-ADP Two hundred milligrams of maximally activated dextran (see Results) were dissolved in 25 ml 0.25 M NaOH; 250 mg ATP or ADP, dissolved in 8 ml of 1.2 M sodium carbonate, was added and the pH immediately adjusted to 9.5 using 1 M HCI. The mixture was stirred at room temperature and 5-ml samples were withdrawn

at specific time intervals. Two approaches were used to block unreacted epoxide groups. In the first, the reaction was stopped in each sample by the addition of 5 ml 2 M sodium thiosulfate; in the second, unreacted epoxide was blocked by the addition of sufficient solid glycine to bring the solution to a concentration of 2 M with respect to glycine. Subsequently, five volumes of cold absolute ethanol were added to each sample, which was then allowed to stand for 12 h at -20°C to ensure maximum precipitation. Studies with unmodified dextran suggested that this corresponds to a recovery of at least 75% ofdextran material. The supernatant was decanted and the precipitates either frozen or redissolved in 10 ml of water for subsequent separation of free cofactor from dextran-bound material. This separation was accomplished either by ultrafiltration or by dialysis. The former method was applied to the entire reaction mixture in which the reaction had been stopped using glycine, as described above. The reaction mixture was loaded into a stirred-tank ultrafiltration cell of 210 ml volume fitted with a 90-mm Amicon PM10 membrane. Distilled water was pumped through at an initial flow rate of 2 ml min-~ and the absorbance of the effluent stream monitored at 254 nm. The absorbance of the effluent returned to zero after 1000 ml had passed through and the cell contents were removed and freeze dried. The dextran-ATP prepared in this way was used for molecular weight studies. Dialysis was used to remove free ATP from samples of the reaction mixture in which the reaction was stopped by adding thiosulfate and precipitating with ethanol. The redissolved precipitates were dialyzed in Visking tubing against 2 I of distilled water for 48 h at 4°C, the dialyzate being changed after 4, 16, 28, and 40 h. The third and fourth dialyzate showed no significant absorbance at 254 nm. The retentate was freeze dried to give solid dextran-ATP. These preparations were used to study the effects of ATP loading on the coenzymic properties of the complex.

Estimation o f dextran content o f dextran-ATP These estimations were carried out on dextran-ATP peparations in which the unreacted oxirane groups had been "sealed" by addition of glycine. The Nelson test for reducing sugars gave a linear response with free dextran but was unsuitable for use with dextran-ATP owing to the formation of insoluble complexes. Refractometry was found to be the method of choice and measurements were made using an Abbd refractometer with a sodium D-line light source at 50°C. Preliminary experiments showed that the molecular refractivity of both dextran T40 and T70 were sufficiently different from that of dextran-ATP to preclude their use as suitable standards. However, as ATP was found to bind to only about 10% of available epoxide groups, it was thought that maximally activated dextran which had been coupled to glycine would be sufficiently similar to serve as a reference. The molecular refractivities of dextran-ATP and dextran-glycine were indeed

Enzyme Microb. Technol., 1990, vol. 12, March

197

Papers found to be virtually identical in the concentration range 0-50 mg ml-I.

Separation o f free A T P f r o m dextran-bound A T P

Enzyme assays

Attempts to separate the free from the bound cofactor by gel filtration on desalting gels (Biogel P2 or Sephadex G25) led in general to incomplete separation of high- from low-molecular weight material. This was almost certainly due to viscosity effects. Elimination of these effects would have required more dilute samples and hence larger sample volumes and correspondingly larger columns leading to difficulties in scale-up; hence this approach was abandoned. Specific adsorption was also attempted by exploiting the known affinity of concanavalin A for the glucose residues of dextran. 18 Affinity chromatography of the crude reaction mixture on Con A-Sepharose 4B was unsuccessful, as it was not found possible to elute the dextran-ATP from the column matrix as a well-defined peak, even using 1 M NaCI or 4 M acetic acid as eluent. Dialysis was the simplest method for removing free cofactor and was ideal for use in preparing laboratory quantities. Analysis of the retentate showed that after 48 h of dialysis as described, less than 2% of the total ATP was present in the free state. Dialysis was also used by Fuller and Bright 2 to purify ATP-substituted polyacrylamides. Ultrafiltration was also effective in removing free cofactor and this may be the method of choice in commercial applications, as the unbound cofactor can be recovered without the extensive dilution necessary for dialysis, thus offering the possibility of reuse. An interesting possibility arises if the bound coenzyme is to be used in an ultrafiitration enzyme reactor, as the reactor itself can be used to prepurify the dextran-ATP.

These were carried out at 30°C using a Cecil 272 spectrophotometer. Kinetic parameters were calculated from direct linear plots.17 Hexokinase activity was assayed by coupling the reaction to glucose-6-phosphate dehydrogenase in 25 mM Tris-HC! buffer, pH 7.7. The reaction mixture contained, in a total volume of 1.0 ml, 50/zmol MgC!2, 100/zmol glucose, 2.0/zmol NAD, ATP, 35/xg hexokinase, and 50/xg glucose-6-phosphate dehydrogenase. Acetate kinase activity was assayed by coupling the production of ATP from ADP to the hexokinase/glucose-6-phosphate dehydrogenase system. The conditions were as for hexokinase assay; in addition, the mixture contained 50/xmol acetyl phosphate and ATP was replaced with ADP. The reaction was started by the addition of 1.2/zg acetate kinase. Determination of enzymically active bound cofactor was carried out as above in 1 ml containing 10/zmol acetyl phosphate and varying concentrations of free or bound ADP. The reaction was started by the addition of 12/xg acetate kinase. All solutions were made up in 75 mM Tris-HC1 buffer pH 7.5, 110 mM MgCI2. The reaction was allowed to proceed for 30 min at 25°C and was stopped by boiling for 3 rain. ATP formed was assayed using the hexokinase assay described above. Control experiments showed that all ATP production had ceased in 30 min and that free ATP was stable at 100°C for 3 rain. Pyruvate kinase was assayed by coupling to lactate dehydrogenase. The mixture, final volume 1.0 ml, contained 0.5/xmol KC1, 20/zmol MgSO 4, 0.5/xmol phosphoenolpyruvate, 0.1 /xmol NADH, and ADP in 80 mM Tris-HCl pH 7.0. The reaction was started by the addition of 6.7/zg lactate dehydrogenase and 5/xg pyruvate kinase. The myokinase assay was based on the hexokinase/glucose-6-phosphate dehydrogenase system described above, with the exception that ATP was replaced by ADP and 50/zg myokinase was used to start the reaction.

Results Activation o f dextran Samples withdrawn at specific times from the dextran activation reaction were assayed as described for oxirane content. Maximal activation is achieved in 45 rain with 220 epoxide groups per dextran molecule. Longer reaction times led to decreasing oxirane content, e.g. at 120 min there were only 50 epoxide groups per dextran molecule. The decantation, rather than centrifugation, of the ethanol precipitates was also important, as centrifugation led to material which was less soluble in water and in which the oxirane content was generally 25-50% of that in the non-centrifuged samples. The concentration of dextran T40 used was similarly optimal. At lower dextran concentrations, the oxirane content of the activated dextran was not significantly increased, whereas higher concentrations led to the formation of insoluble products.

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Estimation o f bound cofactor The amount of cofactor bound per unit weight of complex was determined by two methods. The simplest was to estimate the number of moles present using the known molar coefficients for ATP (15,400 M-I cm -I) and ADP (15,000 M I cm -z) at 254 nm. To ensure that this approach was valid, the results were compared with measurements of the total phosphorus by the method of Bartlett. 19Agreement between the two methods was within 2% for ADP-dextran and 7% for ATP-dextran. These results demonstrate that the extinction coefficient at 254 nm of ATP and ADP is not significantly altered by attachment of the coenzyme to dextran. The agreement between the two methods also indicates that no hydrolysis of terminal phosphates occurred during preparation of the complexes.

Molecular weight estimation The difficulties encountered using gel filtration to "desalt" dextran-ATP indicated that this might not be a reliable method for determination of molecular weight. Viscometry offered a suitable alternative. The intrinsic viscosity of a macromolecule can be related to its molecular weight by an empirical equation of the form

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where K and a are constants for a particular solute/ solvent system and M is the molecular weight. Senti et al. z° found that for dextrans at 25°C, K = 1 × 10 -3 and = 0.5. The intrinsic viscosity of a macromolecule can be determined using a second empirical equation

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To assess the availability of the bound cofactor, the complex was assayed using a hexokinase/glucose-6phosphate dehydrogenase couple, in the presence of excess NAD and glucose. The results, presented in Table 1, show that there is good agreement with ATP content determined by absorbance at 254 nm and by enzymic estimation. The results also show that even at the highest loading achieved, essentially all of the cofactor is available to the enzyme. Table 1 Effect of degree of substitution on cofactor availability to hexokinase

~qsolv

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Number of groups per complex molecule

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Enzyme Microb. Technol., 1990, vol. 12, March

199

Papers 6,0

Specificity of kinases for dextran-ATP and dextran-ADP

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The results described above indicate that yeast hexokinase is able to utilize ATP when the cofactor is bound to dextran. This applies both to samples in which epoxide ends had been sealed with glycine and to those in which the adenine nucleotide coupling reaction had been stopped with thiosulfate. With glycine-stopped dextran-ATP having an average loading of 10 ATP/dextran, the Km value obtained for hexokinase was 0.41 mM as compared to a Km of 0.055 mM for free ATP. The relative V~ax value of bound to free ATP was 5.8. Km and VmaX values for thiosulfate-stopped dextran ATP are described in the following section. Acetate kinase was also found to utilize glycine-sealed dextran-ADP; in this work ADP (free or bound) was incubated with acetate kinase and excess acetyl phosphate until ATP production had ceased. The reaction was stopped and the total ATP produced estimated using hexokinase/glucose-6-phosphate dehydrogenase. The measurements showed that all the bound ADP was available to the enzyme (loading of 10 ADP/dextran). (Similar results were obtained with thiosulfate-sealed dextran-ADP generated enzymically from the corresponding ATP using glucose and hexokinase.) From initial rate studies using a coupled assay consisting of acetate kinase, hexokinase, and glucose-6-phosphate dehydrogenase, the Km of acetate kinase for dextran-ADP was found to be 0.60 raM; K m for free ADP was 0.89 mM under the same conditions. The relative Vm,x (bound ADP/free ADP) was 1.2. Dextran-ADP (glycine sealed) was also coenzymically active with myokinase using a coupled assay system as described for acetate kinase. K m and gma x w e r e not determined. With pyruvate kinase, the results depended on the source of the dextran-coenzyme. Using glycine-sealed dextran-ADP, no activity could be detected in a coupled assay using lactate dehydrogenase to follow the production of pyruvate from phosphoenolpyruvate. However, coenzymic activity was found

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with dextran-ADP (thiosulfate-sealed) which had been enzymically generated by reaction of dextran-ATP with glucose catalysed by hexokinase.

Effect of coenzyme loading o f dextran-ATP on kinetics Kinetic parameters were determined for hexokinase with thiosulfate-sealed dextran-ATP using complexes with average ATP loadings per dextran molecule in the range 18-34. Both Km and Vmaxincrease as the number of ATP molecules per dextran increases. For both kinetic parameters, there appears to be a roughly linear relationship between the parameter and the coenzyme loading (see Figures 3 and 4). Also the values of both parameters are greater than those obtained with free ATP. A similar effect with dextran-ADP and free ADP was seen with myokinase, although the differences in kinetic parameters between the free and bound coenzyme were not so marked.

Stability Activated dextran, stored as a freeze-dried solid at 0°C for 5 months, showed no reduction in the number of free oxirane groups as determined by thiosulfate titration. Dextran-ATP samples, stored either as freezedried solids or dissolved in 25 mM Tris-HCl buffer, pH 7.7 at 4°C, were stable for not less than 18 weeks. This was assessed by the initial rate given by a fixed amount of immobilized ATP in the hexokinase/glucose-6-phosphate dehydrogenase as described. Changes in rate were less than 5% of the initial values.

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Enzyme Microb. Technol., 1990, vol. 12, M a r c h

Reactor runs were carried out to demonstrate the feasibility of continuously recycling the complex. The reactor configuration was based on that described by Gacesa et al. 2~The reactor was loaded with unpurified dextran-ATP and was eluted with molar NaCI until the E254 of the effluent returned to zero. The reactor was

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then washed through with seven residence volumes of the starting buffer, (25 mM phosphate, 150 mM Dglucose, 50 mM Mg2÷ , pH 7.5). The run was started by the addition of 0.35 mg of hexokinase and allowed to proceed for four residence times. At this point the buffer was made 30 m~ with respect to acetyl phosphate and 0.06 mg of acetate kinase was injected into the reactor. The run proceeded for another three residence times before termination. Samples (1 ml) of the effluent stream were collected throughout and were assayed for glucose 6-phosphate; the results obtained are shown in Figure 5.

Discussion The aim of this work was to produce and characterize a soluble dextran-ATP complex using the bisoxirane 1-4-butanediol diglycidyl ether. The method adopted here was to couple adenine nucleotides to dextran T40 using a synthesis protocol based on that used by Sundberg and Porath.15 Initial experiments showed that reaction conditions could be devised to give an activated dextran complex. The critical factor was dextran concentration, which was optimized to prevent the formation of an insoluble precipitate. Work with dextran required a method of separating the activated dextran from unreacted epoxide. Precipitation of the complex with ethanol followed by redissolving in distilled water was considered to be the most suitable method, although the activated dextran is less easily precipitated than the native form. In practice, this problem was overcome by the use of cold ethanol; recoveries in excess of 75% were achieved. Estimation of the number of epoxide groups on the activated dextran was shown to be highly pH dependent. Results were found to be unreliable if fluctuations in pH occurred, and so all estimations were carried out in a pH-stat using a proportional control system. The study of the time course of the activation showed the

reaction to occur over a timescale of 45 min, with further incubation leading to a reduction in the number of groups. It was noticed that at times in excess of 1 h the solubility became markedly reduced. This suggests that, in addition to hydrolysis of epoxide groups, there is some degree of intermolecular crosslinking occurring. To minimize this problem, all subsequent experiments were conducted with the reaction being stopped by reducing the pH to 5.5 prior to precipitation. The time course of these reactions was similar, but the number of active groups was higher. It was also observed that centrifugation of the precipitate accentuated the problem of crosslinking, and this was avoided in later studies. The conditions used for binding cofactor to the activated dextran were essentially similar to those used by Sundberg and Porath 1~for the production of Sepharosecoupled material. Because of the soluble nature of the dextran support, recovery of the product complex is clearly more difficult than for cases where insoluble supports are used. Dialysis, although time-consuming, was found to be the most effective method of purification; however, the use of ultrafiltration membranes was also convenient and this method was used for some subsequent reactor runs, demonstrating that prepurification is not essential. The coenzyme loading on the support showed a maximum value after 12 h. Longer reaction times led to reduction in coenzyme groups bound. The reason for this reduction is not obvious and was not further investigated. The number of cofactor groups was estimated from the published molar extinction coefficients at 254 nm. These values were compared with the measurement of total phosphorus and, when corrected for the number of moles of phosphate per cofactor, gave good agreement with loadings calculated from optical density measurements. Thus the molar E254is not appreciably changed when the molecule is coupled to dextran in this manner. Before the kinetic constants of various enzymes for the complex were measured, the percentage of bound cofactor available to the enzyme was determined. This was found to be 100% for both acetate kinase and hexokinase at the highest cofactor loading obtained. The convenience of this method for producing dextran-cofactor complexes at a range ofcofactor loadings allowed a study to be made of the effect of loading on the kinetic constants obtained with hexokinase. Our results show that both K m and Vmax increase with the degree of loading. Literature reports on the effects of immobilization on apparent K m a r e conficting. Fuller and Bright2 report a decrease in K i n , while other worke r s 9'22'23 report increases compared with free cofactor. Using dextran-ATP prepared from CNBr-activated dextran linked to ATP via a hexamethylene diamine spacer, Yamazaki et al. 9 found, in contrast to our results, that the K mfor hexokinase was unchanged while Vm~xof the dextran complex was 50% that of free ATP. A reduction in Vm~xwas also found with acetate kinase, but the Km for the dextran complex was around 10-fold greater than that for free ATP. Murata et al.]3 studied

Enzyme Microb. Technol., 1990, vol. 12, March

201

Papers the effect of loading on dextran-ATP prepared by Mosbach's 8 method but did not determine kinetic parameters. With both acetate kinase and a glutathione-forming enzyme system, these workers found that enzyme activity fell as loading increased. Increases in Km can be attributed to a range of factors, including steric hindrance 24and a change in chemical environment? The increase o f K mwith loading suggests that steric hindrance is an important factor. As all groups have been shown to be potentially available, the results suggest that not all cofactor molecules can be bound simultaneously. This concept of sterically hindered sites has been used by Wankat 25 to model the performance of affinity-based separations. Thus, although the potential cofactor concentration is equal in each case, the concentration available at any given time is reduced at higher loadings. The increase in Vm,x that we found with increased loading may be explained in terms of the dextran concentration in the assay; i.e. at low coenzyme loadings, higher dextran concentrations are necessary to give the required concentration. To acieve equal cofactor concentrations in the assay irrespective of coenzyme loading requires considerable variation in the dextran concentration. Mosbach 22 has commented on the effect of large coenzyme matrices on gross diffusional mobility. The increased viscosity leads to a decreased reaction rate and would present severe problems in an ultrafiltration reactor. The position of coupling to the cofactor was not extensively investigated. Previous reports have shown that alkylating reagents like epoxides react at the nucleophilic N ~nitrogen of the purine base. The resultant N 1 alkyl derivative undergoes the characteristic Dimroth rearrangement when exposed to hydroxide ions. The evidence for the rearrangement has come from several workers. Fuller and Bright2 demonstrated a characteristic spectral shift from h max 260 nm to 267 nm. Zappelli et al. 26 and Le Goffic et al. 27 confirmed C 6 amino alkyl derivatives using HLNMR spectroscopy. However, in the case of the NADP ÷ derivative prepared by Zappelli et al.,Z6 there was evidence for some side chain modification of the ribose region. The halflife studies of Marcon and Wolfenden28 suggest that not all of the N ~ form would be converted during the reaction time used here. Thus the complex produced might represent a heterogeneous mixture of two or more types of attachment. This was suggested by the qualitative demonstration of the coupling of inosine diphosphate, which lacks a C 6 amino group. From these considerations it is clear that the measured Km for ATP may represent a composite value incorporating the " t r u e " g m value for each individual species. Of the several methods tried, viscosity measurement was the most successful for molecular weight determination. The experimental results gave a figure of approximately 32,000 daltons for dextran T40. This is in accordance with the quoted weight average and number average values for dextran T40. The value obtained for dextran-cofactor complex shows a weight of approximately 80,000 daltons. The sample used in the determination had a measured epoxide loading of ap202

Enzyme Microb. Technol., 1990, vol. 12, March

proximately 200 groups per mole dextran. After 12 h incubation with coenzymes, it was found that 7 molecules of ATP were bound. The remaining epoxide groups were blocked with glycine. Thus the molecular weight of the complex can be estimated from the stoichiometry of the reaction: 1 7 200 193

molecule dextran 32,000 molecules ATP 3,550 molecules spacer 40,450 molecules glycine 14,490 Total ~90,490 Measured value -80,000

In practice, some degree of epoxide inactivation would occur during the incubation with coenzyme; hence the estimate of bound glycine is probably high. Thus these figures give a reasonable agreement with the measured value and suggest that the total complex weight can be approximated if the coupling conditions are known. Attempts to measure concentrations of dextrancofactor complexes using a chemical assay for dextran proved unsuccessful. This was attributed to the masking of reducing groups by the activation procedure; however, it was found possible to measure concentration as a function of refractive index if a suitable defined standard was prepared. The use of these derivatives in enzyme reactors depends on the ability to recycle the coenzyme. It was therefore gratifying to find that the dextran complexes showed activity both with acetate kinase and adenylate kinase. In view of the comparative studies described by Langer, 29 it is important that any derivatized cofactor should show activity with acetate kinase, as this offers the most promising approach to recycling at present. The use of adenylate kinase may be necessary in some situations to maintain an equilibrium between the three adenine phosphates. The utility of the preparations synthesized was demonstrated in experiments using the immobilized cofactor in a membrane reactor. These showed both the feasibility of in situ purification and that the dual enzyme system could successfully recycle the cofactor for at least 60 h.

References 1

2 3 4 5 6 7 8

Weibel, H. K., Fuller, C, W., Stadel, J. M., Buckemann, A. F., Doyle, T. and Bright, J. J. Enzyme Engineering, Vol. 2 (Pye, E. K. and Wingard, L. B., eds.) Plenum Press, New York, 1974, pp. 203-208 Fuller, C. W. and Bright, J. J. J. Biol. Chem. 1977, 252, 6631-6639 Lowe, C. R. Topics Enzyme Ferment. Bioteehnol. 1981, 5, 13-146 Wandrey, C. and Wichmann, R. Biotechnol. Rev. 1985, 5, 177-208 Scmidt, E., Bossow, B., Wichmann, R. and Wandrey, C. Chem. Ind. 1986, 35, 71-77 Furusaki, S., Matsuura, I. and Nazarato, S.-1. Biotechnol. Bioeng. 1982, 24, 1211-1216 Gacesa, P. and Whish, W. J. D. Bioehem. J. 1978,1"/5, 349-352 Lindberg, M. and Mosbach, K. Eur. J. Biochem. 1975, 53, 481-486

Dextran-A TP complexes: J. Hubble et al. 9

Yamazaki, Y., Maeda, H. and Szuki, H. Eur. J. Biochem. 1977,

20

77, 511-520 10 11 12 13 14 15 16 17 18 19

Yamazaki, Y. and Suzuki, H. Eur. J. Biochem. 1978, 92, 197-207 Buckmann, A. F., Morr, M. and Kula, M.-R. Enzyme Eng. 1978, 4, 395-397 Berke, W., Morr, M., Wandrey, C. and Kula, M.-R. Ann. N Y Acad. Sci. 1984, 434, 257-258 Murata, K., Tani, K., Kato, J. and Chibata, I. J. Appl. Biochem. 1979, 1, 283-290 Suzuki, H. and Yamazaki, Y. Methods Enzymol. 1987, 136, 46-55 Sundberg, L. and Porath, J. J. Chromatogr. 1974, 90, 87-98 Axen, R., Drevin, H. and Gaulsson, J. Acta Chem. Scand. B 1975, 29, 471-474 Eisenthal, R. and Cornish-Bowden, A. Biochem. J. 1974, 139, 715-720 Sharon, N. and Lis, H. Science 1972, 177, 949 Bartlett, G. R. J. Biol. Chem. 1959, 234, 459-465

21 22 23 24 25 26 27 28 29

Senti, F. R., Hellman, N. N., Ludwig, N. H., Babcock, G. E., Tobin, R., Glass, G. A. and Lamberts, B. L. J. Polymer Sci. 1955, 17, 527-546 Gacesa, P., Eisenthal, R. and England, R. Enzyme Microb. Technol. 1983, 5, 191-195 Mosbach, K. Adv. Enzymol. 1978, 42, 205-278 Mosbach, K. Methods Enzymol. 1987, 136C, 1-9 Schmidt, H. L. and Grenner, G. Eur. J. Biochem. 1976, 67, 295-302 Wankat, P. C. Anal. Chem. 1974, 46, 1400-1408 Zappelli, P., Rossodivita, A., ]5"osperi, G., Pappa, R. and Re, L. Eur. J. Biochem. 1976, 62, 211-215 Le Goffic, F., Sicsic, S. and Vincent, C. Eur. J. Biochem. 1980, 108, 143-148 Marcon, J. B. and Wolfenden, R. Biochemistry 1968, 7, 3453-3458 Langer, R. S., Hamilton, B. K., Gardener, C. R., Archer, M,C. and Colton, C. K. AIChE Journal 1976, 22, 1079-1090

Enzyme Microb. Technol., 1990, vot. 12, March

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