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Immobilization of aspartate aminotransferase on agarose
Bit,chimic 71 (1989) 439- 448 (~ Societd de ('himie biologique / Elsevier, Paris
439
Immobilization of aspartate aminotransferase on agarose Kalevi KURKIJARVI* and Timo K O R P E L A
Department of Biochemistry, University of Turku, SF-20500 Turku, Finland (Received 11-8-1988, accepted after revision 24-11 - 1988)
S u m m a r y ~ Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg / ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg / ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vm~,xwas 51%. Chcr,,ical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 m g / m i of the gel). The apparent specific activities were 27-35°/~ while the value calculated from Vmaxwas 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vm~xto 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined. aspartate aminotransferase / agarose / immobilization methods / active site / bromphenol blue / pyridoxal 5'phosphate
Introduction Vitamin B6-dependent enzymes are involved in the central metabolic routes of amino acids and biological amines. A large variety of these enzymes, differing in their catalytic properties, exist in nature. Vitamin B6-enzymes have important applications in large-scale production of amino acids from synthetic precursors [1]. Side effects of PLP-dependent enzymes may also be used in the production of synthetic compounds such as refined chemicals and pharmaceuticals. Recent developments in site-directed mutagenesis for altering specificities with increased capability for producing the enzymes by recombinantDNA methods should give new impetus to the search for practical uses in industry and analytics for this class of enzyme.
In any practical applications of B~-enzymes, the possibility of immobilization on a solid support should be considered because of advantages such as improved stability and reusability [2]. With vitamin-B6 enzymes, this immobilization may be of special importance as many of these enzymes are unstable. On the other hand, their active site contains pyridoxal 5'-phosphate with other catalytic functions that are reactive towards accessible reagent [3], which necessitates proper selection of coupling techniques. This property largely exciudes the coupling reactions that employ activation of the enzyme itself. The present study was conducted to find means of immobilizing PLP enzymes by using cytosolic aspartate amino-transfcrase (AspAT) from pig heart. This enzyme is the most widely studied among the PLP enzymes and details of
*Present address: Wallace Oy, Biochemical Laboratory, P.O. Box 10, 20101, Turku, Finland. Abbreviations: AspAT: Aspartate aminotransferase; EC 2.6.1.1: glutamic-oxaloacetic aminotransferase; PLP: pyridoxal 5'-
phosphate; PMP: pyridoxal 5'-phosphate.
440
K. Kurkijgirvi and T. Korpela
its mechanism and structure have been well documented [3]. Various aspects of the immobilization of AspAT from Escherichia coli on to glass and diatomaceous earth have been recently reviewed by Rozzeli [4].
Materials and methods Chemicals L-Aspartate, L-cysteine, cacodylic acid, malate dehydrogenase (15/~kat / rag), Coomassie brilliant blue G-250, alpha-ketoglutaric acid, pyridoxal 5'-phosphate, and pyromellitic acid were purchased from Sigma Chemical Co., St. Louis, Mo. Phthalic acid, bromphenol blue, and 4-aminobutyraldehyde diethylacetal were from E. Merck A.G., Darmstadt. Glutaric acid, maleic acid, and oxaloacetic acid were from Fluka A.G., Buchs and cyanogen bromide was from Aldrich Chemical Co., Milwaukee W.I. Sepharose CL 4B and activated SH-Sepharose 4B were delivered by Pharmacia Fine Chemicals, Uppsala. The Radiochemical Centre (Amersham, UK) supplied N-ethyl(2,3- C)-maleimide (10mCi / nmol). Bioluminescent NADH Monitoring Kit was kindly donated by Wallac Oy, Turku. The alpha-subform of cytosolic aspartate aminotransferase was purified from pig heart according to Martinez-Carrion et ai. [5]. The specific activity of the AspAT preparations was at least 2.4 p,kat / mg. Immobilization procedures The butyraldehyde derivative of Sepharose CL 4B was synthetized according to Korpela and Hinkkanen [6]. Two g of moist aldehyde-agarose was suspended in 2 ml of 0.1-0.01 M potassium phosphate, pH 7.0-9.0 containing AspAT 0.5- 10.3 mg. The suspension was gently shaken for 20 h at 4oC. The enzyme-gel conjugate was first washed with 50 vol. of 0.01 M potassium phosphate, pH 7.0, containing 1 M KCI and 0.5 mM PLP, followed by 100 vol. of the same buffer without PLP. The azomethine linkages between the gel and enzyme were reduced at 4oC by adding 250/.tl of cyanoborohydride in methanol (1 m g / m l ) after a 20-h incubation of the gel and enzyme in the buffer as above, supplemented with 30% methanol followed by a 30-min incubation with the reducing reagent. The resulting gel was washed with 10 vo! of 30% methanol and 10 vol of water
and finally with 10 vol of 0.1 M potassium phosphate, pH 7.0. The gels were stored at 4°C in 2 ml of the final washing buffer. Activated SH-Sepharose 4B was treated according to the manufacturer's directions. One ml of the swollen gel (0.35 g of the lyophilized powder) was suspended in 2 vol of 0.1 M potassium phosphate, pH 7.0, containing 0.1 M KCi, 1 mM EDTA with AspAT. The suspension was gently shaken at 4°C for 20 h and the gel washed as after CNBr-coupling (see below). The enzyme was released from the support (1 ml) with 50 mM potassium phosphate buffer, pH 8.0, containing 5 mM L-cysteine [7]. Enzymatic activity and protein were measured after overnight dialysis at 4oC against the same buffer containing 0.5 mM PLP. Sepharose CL 4B (2 g of moist, suction-dry gel, equal to 2 ml bed vol) was activated with CNBr by the buffer method (200 mg of CNBr per activation unless otherwise stated) as described elsewhere [8]. The washed activated agarose was normally suspended in 4 ml of 0.2 M potassium phosphate, pH 7.0, containing the enzyme. The suspension was gently shaken at 4oC for 20 h. The resulting AspAT-agarose was first washed with 5-fold vol of 0.01 M potassium phosphate supplemented with 0.5 mM PLP and with 1 mM glycine (pH 7.0), and then with 5-fold vol of 0.01 M potassium phosphate supplemented with 1 M KCi and 0.5 mM PLP (pH 7.0) followed by wash;H. ~. . . . w .i~-h ~ h , a ~ r c t ~ n, H s e c o n d ~a~mch u ~ ~ f l ,,,-~i v~.,, ,.,~z h a t h ing solutions. The suction-dry enzyme-gel was suspended in 2 ml of 0.1 M potassium phosphate, pH 7.0, containing 0.5 M KCI and stored at 4oC. ,,JlU?
I~IFJI+ l , . J l k . , t t r L l l
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III~II.
~&
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Determination of protein and enzymatic activity Soluble proteins were measured with the biuret assay against bovine serum albumin, and the Coomassie brilliant blue [9] method (for dilute solutions) was standardized against it. Sepharose-bound protein was calculated from the differences between the soluble protein before and after the coupling process. Except for the studies carried out with SH-Sepharose, proteins were also determined by N[t4C]-ethylmaleimide-labeled AspAT added to the enzyme to be coupled [ 10]. These 2 methods correlated well; the radioactive method usually showed only a few percent less bound protein concentrations. The AspAT enzymatic reaction was measured kinetically from alpha-ketoglutarate and Laspartate (both 20 mM unless otherwise mentioned to oxaloacetate and glutamate at 25°C in 0.1 M potassium phosphate, pH 8.0. Enzyme activ-
Immobilized aspartate ami~,u;~rat!,:ferase ity was expressed as micromoles of oxaloacetate formed per second (v&at). Soluble enzyme was normally measured at a protein concentration of 1 g g / m l . The immobilized enzyme was measured with a stirred batch reactor [11, 12] using 20 mg of suction-dry enzyme-gel in the reaction vol of 13 ml. Enzyme-bound PLP was measured fluorometrically after release of the coenzyme with 5.5% ( w / v ) trichioroacetic acid at 50°C. Bioluminescent determination of the active centers and maximum rate calculations are presented elsewhere [ 12].
Results
Butvraldeh y d e - agarose Cy(osolic pig heart aspartate aminotransferase contains 19 lysine residues per subunit [13], part of which could react with aldehyde groups on solid support through the Schiff base reaction under mild chemical conditions. AspAT retained its activity very well, buc coupling efficiency was low (Table I). The latter result was obviously due to the fact that the Schiff base linkages were formed reversibly and the enzyme therefore leaked out of the gel during extensive washing. Coupling efficiency increased at low iotfic strength (10 raM) and at higher pH (9.0) of the reaction and washing buffers, but AspAT was uetacucu immeuiately when higher salt was used or the pH was lowered. Ion-exchange interactions were probably responsible for binding of this elutable protein. Although the percent coupling was low (Table I), it could be seen that a small part of the enzyme was stably bound and was not lost during extensive use under reactor conditions. High apparent (observed) specific
441
activity was due, in part, to the low bound enzyme activity resulting in low diffusional limitations. Fixation of the imino bonds by reduction with cyanoborohydride ;~ a routinely used method for stabilizing them [14]. !n ,the case of PLP-enzymes the reduction may noz be favorable because of the subtle reduction in the PLP-apoenzyrne Schiff base linkage with concomitant loss of catalytic activity [3, 4]. In a test in which the Schiff base linkages were reduced, the coupling efficiency increased 10-fold with complete loss of activity. This confirmed the presence of a substantial amount of sterically available reactive aldehyde groups on the gel. Immobilization c f A s p A T on cvanuric chloride- and divinyl-sulfone-activated agaroses [15] was tested. In both cases only 3 - 5 % of activity was found after immobilizati,3n. The low figures can be explained by the poor elutability of the activating compounds from the gels.
Immobilization on thiol-agarose AspAT contains 5 SH-functions per monomeric enzyme molecule, 3 of which can be modified with soluble reagents, whereas the final 2 cysteinyl residues react only under extensive denaturing conditions [16]. Immobilization on SHfunctions is of special interest since the enzyme can be detached with soluble thiols and the resulting enzyme comp:~rocl u, ith th~ ,~,4,,,;,,~I ,,,a,~,identical reaction conditions. Coupling of AspAT to SH-agarose was incomplete at any concentration, while maximal binding capacity was ~3 m g / m l of gel (Fig. 1)o Apparent bound activity was found to be linearly dependent on the bound protein up to =1.5 m g / m l of gel with an enzyme preparation with asp. act. of 3/~kat/rag. The apparent spe--
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Table I. Immobilization of AspAT on butyraidehyde-agarose. Protein (rag)
Activity (#kat)
Coupled protein (mg/ml)
Coupled activity (/.tkat / ml)
Apparent S.P. Act. (/xkat / mg)
I).5 1.0 2.6 5.1 10.3
1.2 2.4 6.3 1'2.3 24,9
(1.02 0.04
0.03 0.05 0.09 0.15 0.25
1.50 (63) 1.25 (52) 1.29 (54) 1.50 (63) 1.19 (50)
0.07
0.10 0.21
AspAT was immobilized without cyanoborohydride reduction as described in Materials and methods. The specific activity of the AspAT preparation was 2.4 ttkat/mg. The proteins were measured by radioactively labeled enzyme. The percentage specific activitiescompared to the solu01eenzyme are shown in parentheses.
K. Kurkijiirvi and T. Korpela
442
-z""0
of enzyme activity during the procedures were taken into account practically all of it was recovered in the soluble form. No significant differences in the immobilization properties of the gel were observed when immobilization was repeated 3 times. The enzyme itself did not denature upon immobilization (see Fig. 3 and above). One obvious explanation for the apparent loss of activity is the mass transfer barriers provided by the
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Fig. 1. Capacity of S H - S e p h a r o s e to bind AspAT. The solution of immobilization contained 3 ml of 0.1 M potassium phosphate, pH 7.0, 0.1 M KCI, 1 mM EDTA, 1 ml of t h e ool
anti 1] 3 - - 0 ~ . . . . . . . . . . . . . .
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3.0 /zkat/mg). The reaction was allowed to proceed for 20 h at 4°C. (a) bound protein; (b) apparent bound enzyme
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Fig. 2. Binding kinetics of AspAT on SH-Sepharosc. The immobilization took place in 24 mi of potassium pht)sl~hatc, pH 7 0 containing 0.1 M KCI with 8 ml of moist gel and 24 mg of AspAT (specific activity 3.(1/xkat/mg) at 4"C. Samples were assayed from the su,,;pension at appropriate time intervals. The results are expressed percentually of the soluble enzyme at the beginning of the procedure in the immobilization solution when bound protein and activity is 0 % and the supernztant protein and activity is 100%.
activity.
% OF :[[41TI,.:iL qL':TIVIT"/
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cific activity was 3 0 - 4 0 % of the soluble one in the valid range of enzyme concentrations. The time-course of coupling of AspAt on SH-agarose (Fig. 2) was similar to the reaction rate of soluble reagents [16]. A tendency to 3 different reaction steps was found (Fig. 2). Protein binding and activity correlated well, suggesting that the reaction product was equal with respect to activity remaining within the advance of the reaction. A preparation of immobilized AspAt containing 2.8 mg protein / ml of the 8c! was eluted from the gel with 5 mM cysteine [7]. After dialysis against buffer containing 0.5 mM PLP, the recovered percent ot protein was 93 while that of enzyme activity was 83. The immobilized gel did not show any activity after cysteine washing with PLP-containing buffer. When normal losses
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Fig. 3. Thermal stability of SH-Sepharose-immobilizcd AspAT. The immobilized preparation ( I mg AspAT / ml of gel) was incubated at 70,'C in 20 mM potassium phosphate, pl! 8.0 (15 mg of moist gel in 2.5 ml of the buffer) containing eithel ! mM L-aspartate or 1 mM alpha-ketoglutarate. The enzyme reactivated in the presence of i,-aspartate (PMPform) was reactivated by incubating the enzyme in 20 mM potassium phosphate, pH 8.0, containing 10 mM PLP at 5"C with gentle shaking.
Immobilized asparlate aminotransferase gel network. They can be minimized by reciprocal measuring of the apparent activities at high substrate concentrations [12]. In this manner Vm~xof 51% of the soluble enzyme was achieved (Table II). Since the Vmax values were nearly constant and the Km values reasonable compared with the soluble enzyme, at least up to ~1 rag, enzyme p r o t e i n / m l of gel (Table II), the Vm~ value for bound A s p A T (51%) was reliable. Hence, the 49% reduction in enzyme activity most probably resulted from modification of thiol groups on AspAT, While the half-life of the enzyme activity of A s p A T - S H - a g a r o s e at 4°C in 0.1 M potassium phosphate, pH 7.0 supplemented with 1 M KCI was ~ 2 y, that of soluble enzyme was 2 mo. Good storage stability, with distinctly increased thermostability of the immobilized A s p A T (r~/2 = 10 h at 70oC) compared with soluble AspAT in the PLP form (rl/2 = 2 h), suggested that at least one covalent bond to each monomeric unit existed. When the enzyme was transformed to the PMP form with aspartate [12], the half-life of inactivation at 70"C was ~5 min (Fig. 3), which was exactly the same as with the soluble enzyme; i.e., immobilization did not protect ,L~,,,,:PMP form of the enzyme. The enzyme was, bowever almost completely reactivatable flora the stage of 50% inactivation with PLP treatment (Fig. 3), whereas in the absence of PLP no reactivation appeared.
443
CNBr-activated Sepharose Binding capacity and binding strength in immobilization can be controlled both by varying the activation degree of the gel and by the pH of the immobilization reaction. Both these factors depend on the properties of the enzyme and on the pH-reactivity profile of the support. The immobilization reaction was carried out at pH 6.0, 7.0, and 8.0, at different degrees of activation. Figure 4 shows that protein binding capacity was about the same at pH 7.0 and pH 8.0, but the observed activity of the preparation bound at pH 7.0 was almost ?-,Cold higher than at pH 8.0. Immobilization at pH 6.0 showed lower protein coupling and remaining activity than at neutral pH (Fig. 4). The apparent optimum for CNBr concentration in the activation was ~50 m g / ml of the gel (Fig. 4) with the remaining activity used as the criterion. HoweveL the result was no longer optimal for thermostability of the preparation (Fig. 5). For further experiments, activation with 100 mg C N B r / m i of agarose was chosen as a compromise. The capacity of the CNBr-activated agarose to bind A s p A T was tested by varying the amount of enzyme in the immobilization. Bound protein increased in a strictly linear fashion as a function of the enzyme, and no saturation concentration was reached up to 9 m g / m l (Fig. 6a). On the contrary, an apparent saturation of bound
Table il. Comparison of the Km values for r-aspartate (KmA) and for alpha-ketoglutarate (KrnB) and the Vmax
velocities for immobilized and soluble AspAT. A Km (mM)
B Km (mM)
Vmax (p,kat / mg)
Soluble AspAT
4.(I
0.8
3.5
AspAT-Stt-agarose 0.30 mg / ml 0.60 mg / ml 1.00 rag/ml 2.81) mg / ml
4.4 4.9 5.4 6.5
1.7 1.9 2.6 5.0
1.8 1.8 1.7 1.4
AspAT- CN Br-aragose 0.35 m g / m l 1.30 mg/ml 2.70 rag/ml 4.00 mg/ml
2.5 3.3 4.2 4.1
1.5 1.9 2.4 2.4
1.6 1.7 1.4 1.2
Enzyme preparation
The concentration of soluble AspAT (specific activity 2.4-3.11/~kat/mg) was 1 /~g,/ml and immobilized enzyme was used 11)-25 mg of moist gel in the reaction volume of 13 ml [12].
K. Kurki#irvi and T. Korpela 4 SC~J,'4DFP.OTEIH c.rL=.:, a mlm~UlI~UlUlUIIIIIIIIIII
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Fig. 4. The effect of the number of reactive groups on CNBractivated Sepharose on AspAT immobilization at different pHs. With each immobilization, 3.6 mg of AspAT (specific activity 2.4 t~kat / mg) were employed. Immobilization was carried out in 0 2 M n• n t a K ~ i H m nhncnhat~ hi--I[ K f l '7 f l n r ~ i~=. v.v~ , .v~ vl 8.0. (a) boun-d protein; (b) bound apparent enzyme activity. . . . . . . . . . . .
BOUND PROTEIH (mg,r~,L,:,*,_=el:."
8.0
2oo
15o
CNBr/GEL (mg4E;,
. . . . . .
enzyme activity was reached at about 3 m g / m l (Fig. 6b). Immobilization of proteins has been reported to occur on CNBr-activated agarose within 2 - 3 h at room temperature and within = 2 0 h at 4°C [17]. Kinetics of immobilization on to CNBractivated agarose was studied at 4°C by measuring the specific activities of the enzyme in the coupling buffer and in the gel-bound stage (Fig. 7). More than 90% of protein was bound in 6 - 8 h; however, no increase in the apparent enzyme activity was found after 2 - 3 h (Fig. 7). A special feature of the experiment shown in Fig. 7 was that enzyme activity decreased faster than the bound activity increased. This finding prompted us to study further the effect of the washing time of the gel, since it was conceivable that the enzyme inhibition was caused by free CNBr in the solution. It has been recommended
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o
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AzpAT !!-IINNOE',ILIZATIOHBUFFER c:rYLl.'r~,L) BOUND ACTIVITY (ukat/mL)
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TINE (hours)
Fig. 5. Thermal stabilities of AspAT's immobilized at different CNBr-activation degrees. The conditions and preparation of the experiments were as in Fig. 3. The soluble enzyme was treated in the same conditions at concentration of 0.15 mg/ml.
Fig. 6. Binding of AspAT on CNBr-activated Sepharose. Immobilization was carried out at 4°C (20 h) in 6.0 ml of 0.2 M potassium phosphate, pH 7.0, containing 2 ml of moist AspAT-agarose carrying 0.3-9.0 mg of enzyme (specific activity 2.4 /xkat/mg). (a) bound protein; (b) apparent bound enzyme activity.
Immobilized aspartate aminotransferase in the general procedures for CNBr activation that washing and transfer to the coupling solution be done within <2 min to obtain maximum coupling efficiency [17]. We found that when washing time was increased from 1.5 min (1.5 m l / s to 5 - 7 min, the observed activity of the immobilized AspAT was 10% U higher than with the shorter washing time, while the coupling efficiency of the protein remained the same. Washing for <1.5 rain clearly showed that either unreacted CNBr or another active compound released from the activated gel was responsible for the enzyme inhibition. The latter
445
alternative was also implied by the prolonged decrease in the specific activity of the soluble enzyme (Fig. 7, compare with Fig. 2). The apparent specific activities of immobilized AspAT preparations were from 27% to 35% in the region of valid enzyme ioadings extending up to ~ 1.5 m g / m l of gel. According to the measured V~ax values (Table II), the specific activity yield of 46% was obtained as calculated from the smallest protein loading. As with SH-Sepharose, the Km values were related to the soluble enzyme with slightly lowered Km for aspartate and increased for 2-ketoglutarate.
Protection of AspA T against activity losses OF USED I00 i I,AHt i i~ I~ i I H II # nl 0 l,~ i b ill i , I lm I ::
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, - , BOUHD ACTIVITY ---BOUND PROTEIH SP. ACTIV.
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soluble 0
-
4
6
R
IHt4OBILIZATIOH TItlE (hours)
Fig, 7, Binding kinetics of AspAT on CNBr-activated agarose. Immobilization took place at 4°C in 22 m! of 0.2 M potassium phosphate, pH 7.0, containing 11.5 mg of AspAT (specific activity, 2.4/.tkat/mg). The results are expressed in percent of the soluble enzyme at the beginning of the coupling procedure (see text in Fig. 2).
Since AspAT seemed to be sensitive to inactivation by soluble reagents during the immobilization procedures, potentially protective compounds in the coupling buffer were studied (Table III). The coenzyme PLP did not show any effect on immobilization results, indicating that coenzyme loss did not occur during immobilization. A slightly lower activity yield was actually observed with glutarate and maleate. In the presence of more bulky aromatic carboxylic acids, pyromellitic acid and phthalic acid, a reduction in activity was also obtained. Specific activity was slightly improved with slightly lower binding of the protein in the presence of both substrates. Thi,s may, however, be an artefact because o'~ reacts to the gel through its amino aspart,~,~, group and thus the result reflects the effects of a less reactive gel. Cacodylate and bromphenol blue significantly
Table I i l , Effect of active site-protecting c o m p o u n d s on immobilization of A s p A T on CNBr-activated agarose.
Additive
Bound protein (rag/ml) (%)
Bound sp. activity (~kat / m,~) (%)
None
1.5
(94)
(I.65
(27)
10 mM glutarate 10 mM maleate 10 mM pyromellitinic acid l0 mM phthalate l0 mM L-aspartate-ketoglutarate 0.5 mM PEP 0.01% Bromphenolblue ' 0.2 M cacodylate
1.5 1.4 1.4 1.4 1.3 1.2 1.4 0.6
(94) (88) (88) (88) (81) (75) (88) (38)
0.45 0.38 0.58 0.27 0.85 0.65 1.44 1.22
(19) (16) (24) (1 l) (35) (27) (60) (51)
The specific activity of soluble AspAT was 2.4/.tkat / mg which was used at a concentration of 1.6 m g / ml of gel. The immobilization was performed in 0.2 M potassium phosphate (0.2 M, pH 7.0), except in the case of potassium cacodylate which replaced the potassium phosphate (pH 7.0). The specific activities were measured in standard conditions involving 20 mM substrates. The percentage coupling efficiences are presented in parentheses. Bound protein was determined with radioactively labeled AspAT.
446
K. Kurkijiirvi and T. Korpela
increased the immobilized specific activity (Table III). Of the two, bromphenol blue was more favorable because it did not affect the coupled protein. Maximal protection by bromphenol blue was obtained at 0.01% ( w / v ) concentration (Fig. 8). Table IV shows Vm~, values for AspAT with measured PLP content, transaminable active sites, and turnover rate for the soluble enzyme that immobilized conventionally and that which immobilized in the presence of bromphenoi blue. The Vm~,xof the preparation immobilized in the presence of bromphenol blue was almost twice that in its absence. The coenzyme was not significantly lost in either case. The number of active transamination centers drastically increased in the presence of bromphenol blue, while their average turnover increased only slightly (Table IV). Because added PLP did not shield the enzyme from activity loss (Table III) and because the measurable PLP content with and without bromphenol blue was practically identical (Table IV), the free CNBr reacted to the active site to a function other than PLP. This was also supported by immobilization of the PMP form of AspAT which showed the same remaining activity as the PLP form procedure.
Discussion In the 1970s a number of studies were published on AspAT immobilization, lkeda et ai. [18] showed that the apoenzyme is bound similarly to the holoenzyme on CNBr-activated agarose, indicating that iysine-258 is not reactive. Immobilized AspAt was used to measure L-aspartate in micromolar quantities [18]. In addition, AspAT was immobilized on aminoethyl cellulose [19] and on collagen film [10, 20, 21] and immobilized
BOUNDSP. RC~IVITY (ukatx~g)
1,5
1,0
0.5 0
0.:]04
0.008
0.012
BROMPHEHOLBLUE (%P Fig. 8. Effect of the concentration of bromphenol blue on AspAF immobilization on CNBr-activated Sepharose. The conditions of the experiments are similar to those in Table II1.
AspAT was used as a kinetic model of 2-substrate enzyme reaction. Arrio-Dupont and Coulet (1979) studied AspAT subunit interactions by using collagen film-immobilized enzyme and showed that only the dimeric enzyme is active [22]. Woven silk was used as the support for immobilized AspAT by Grasset et al. [23]. In the 1980s, events taking place during the immobilization of AspAT on CNBr-activated agarose were studied in detail [12], and later, immobilized AspAT was used coupled to bioluminescence enzymes to measure L-aspartate and oxaloacetic acid at the picomole level [25]. Recently interest in the immobilization of AspAt seems to have increased with promising results en the utilization of side effects of AspAT for production of certain metabolites [4, 24]. Although immobilization efficiency on aldehydic agarose containing 4-carbon spacer was
Table IV. C o m p a r i s o n of properties of soluble A s p A T with c o n v e n t i o n a l l y immobilized A s p A T 1 and A s p A T
immobilized in the presence of 0.01% (w/v) Bromphenol blue (AspAT 2). Parameter
Soluble
Bound AspAT 1
Bound AspAT 2
Vm~,x(p.kat / rag) PEP (nmol / rag) Transaminationable active centers (nmol / mg) Turnover number (~kat / nmol)
3.08 12.8
1.26 (41) 11.0 (86)
2.46 (80) 11.5 (90)
12.8 0.24
6.7 (52) 0.19 (79)
11.0 (86) 0.22 (93)
The .percentage values compared to the soluble enzyme are shown in parentheses. The turnover numbers were calculated as the maximum velocity per "'transaminationable" active center.
hnmobili: od aspartate aminotrans]erase low, this method yielded the highest operative specific activities (Table I). Glutardialdehyde coupling and dialdehyde supports achieved by periodate oxidation have often been used for immobilization of enzymes with borohydride reduction [15]. These reactions are complex and also involve reactions other than the Schiff base reaction. The method employed here was more straightforward and novel in ~he context of protein immobilization and the results show that a small portion of the enzyme was firmly bound to the gel, although the Schiff base linkage, as such, may not be stable enough. Thus, the mechanism of binding must involve a multipoint attachment of the enzyme at specific microenvironments on the gel. A large number of aldehyde functions remained free as proved by a 10-fold increase in bound protein in the presence of cyanoborohydride. The aldehyde matrix employed could also serve as a special chromatographic material capable of separating proteins and other substances according to the number of amino functions. S H - a g a r o s e can be considered as a modifying reagent; compared with the soluble reagents it has steric hindrances which may be utilized in studies of the SH groups located on the surfaces of proteins. Cysteines 45 and 82 of cytosolic AspAT react readily with soluble thiol reagents. Within their modification, activity varies between 5 6 - 1 2 5 % of that of the original enzyme [26]. The third modifiable residue, cysteine .~,v, ~"~" r e a c t s , syncataiyticaiiy, in the presence of substrates with an activity decrease to < 5 % , reflecting conformational movements necessary for catalysis [16, 26, 27]. Since the remaining activity of the support-bound enzyme was ~50% and since the enzyme was immobilized in the absence of substrates, cvsteine 3911 scarcely took part in the process. An3, slow reaction to cystein-390 did not occur because the preparation was stable over extended periods. Against the background of the cited modification studies by soluble reagents, the present loss of enzyme activity upon immobilization most probably resulted from decrease of flexibility of the enzyme molecule when bound through cysteinyl residues 45 a n d / o r 82. Immobilization on SH-agarose did not protect the PMP form of AspAT from denaturation as compared with the soluble enzyme. Fig. 3 shows that denaturation commences with a reversible release of PMP followed by more or less reversible denaturative stages of the apoprotein. Because the enzyme was bound through at least
447
2 covalent bonds to the solid matrix, dissociation into subunits was not involved in the procedure. Conversely, the results suggest that denaturation of soluble enzyme follows the same mechanism. The lack of protection of the PMP form of AspAT by immobilization also supports the argument that the enzyme was bound to the support through SH functions located relatively far from the active sites. Chromatographic behavior of heat-treated AspAT has shown that while enzyme activity remains unaltered, irreversible changes in the apoprotein can occur in the enzyme [28]. Hence denaturation of the active site region and of other parts can occur independently. A remarkable property of immobilization on CNBr-activated agarose was that the remaining activity was very sensitive to the washing time of the activated gel. The present results show that successful immobilization of vitamin B6-dependent enzymes requires extraordinary care when gel-activating reagents are removed. With other enzymes, traces of the reagents may not impair the yields, but with B6 enzymes this is not the case. Hypotheses about free CNBr and another solubilized reagent (e.g., trimerized CNBr) in the ceupling buffer led us to study active siteprotecting compounds. The most significant finding was that bromphenol blue dye was a very effective agent, increasing the remaining activity ~2-fold compared with conventional coupling with moderate whashing time of the gel (Table IV). Bromphenol blue was effective in concentrations of about 3-fold the molarity of enzyme. When one considers probable binding of the dye on the agarose matrix, binding to the enzyme was nearly quantitative. The shielding was obviously due to the !act that bromphenol blue binds reversibly at or near to the active site with Kl of 0.6 x 10- ~'[291. Other mono- and dianions, including cacodylate, also bind pH-dependently to AspAT [30]. The dicarboxylate acids tested (Table III) decreasfd the activity rather than increasing it, which was somewhat'surprising for they are known to induce a - closed conformatioil ,, of the active site [3] and it was conceivable that this would prevent reactions near the active site. Because bromphenol blue and cacodylate are both anionic compounds at pH 7.t), they may protect by binding to the same positively charged function and thus only one catalytically important function may be the subject of protecflop. These observations show that active-site protecting compounds can be found among known reversible inhibitors of the enzyme.
448
K. Kurkijiirvi and T. Korpela
The calculated average turnover rates and the K m values (Table II) showed that the properties of the enzyme changed only slightly upon immobilization, suggesting that the active sites were not embedded in gel, thus the chemical milieu was not essentially different from the soluble reaction. While bromphenol blue clearly protected against soluble reagents, it also slightly increased the average turnover rate (Table IV), which shows that it shielded the enzyme from modification by the solid reagent. The active site-protecting agents of the present study were quite different from other studies with AspAT from E. coli [4]. Our attempts to employ carbodiimide reaction of coupling yielded inactive enzyme, whereas E. coli enzyme has been successfully immobilized by carbodiimide in the presence of substrates and PLP [4]. On the other hand while A s p A T retained its activity when immobilized on S H - a g a r o s e well, alanine aminotransferase completely lost activity in the process (unpublished observations). These aspects show that enzymes catalyzing similar reactions can behave quite differently upon immobili~:ation, as is the case with modifications by soluble reagents. As stated by Rozzell [4], a common feature of transaminases and other vitamin B6-dependent enzymes seems to be dependence on suitable immobilization chemistry.
References 1 Soda K., Tanizawa K., Esaki N. & Tanaka H. (1987) in: Biochemistry of Vitamin B6; Proceedings of the 7th International Congress on Chemical and Biological Aspects of Vitamin B6 Catalysis (Korpela T. & Christen P., eds.), Birkh/iuser Verlag, Basel, Boston, pp. 435-444 2 Lilly M. & Dunnill P. (1976) in: Methodsin Enzymology (Mosbach K., ed.), Academic Press, London, vol. XLIV, pp. 717-738 3 Arnone A., Christen P., Jansonius J. & Metzler D. (1985) in: Transaminases (Christen P. & Metzler D., eds.), John Wiley, New York, pp. 326-362 4 Rozzell J. (1987) in: Methods in Enzymology (Mosbach K., ed.), Academic Press, London, vol. 136, pp. 479-497 5 Martinez-Carrion M., Turano C., Chiancone E., Bossa F., Giartosio A., Riva F. & Fasella P. (1967) J. Biol. Chem. 242, 2297-2409 6 Korpela T. & Hinkkanen A. (1976) Anal. Bio-
chem. 71,322-323 7 Carlson J., Axen R., Brocklehurst K. & Crook E. (1974) Eur. J. Biochem. 44, 189-194 8 Korpela T. & Kurkij/irvi K. (1980) Anal. Biochem. 104, 150-152 9 Bradford M. (1976) Anal. Biochem. 72,248-254 10 Coulet P., Godinot C. & Gautheron D. (1975) Biochem. Biophys. Acta 391,272-281 11 Kurkijarvi K. & Korpela T. (1981) Biotechnol. Bioeng. 23, 1389-1392 12 Kurkij~irvi K. & Korpela T. (1983)Appl. Biochem. Biotechnol. 8, 135-144 13 Barra D., Bossa F., Doonan S., Fahmy H., Hughes G., Martini F., Petruzzelli R. & Wittman-Liebold B. (1980) Eur. J. Biochern. 108, 405 -414 !4 Parikh I., March S. & Cuatrecasas P. (1974) in: Methods in Enzymology (Jakoby W. & Wilchek M., eds.), Academic Press, London, vol. 34, pp. 77-102 15 Turkova J. (1978") in: Affinity Chromatography. Elsevier, Amsterdam, pp. 153-189 16 Birchmeier W., Wilson K. & Christen P. (1973) J. Biol. Chem. 248, 1751-1759 17 March S., Parikh I. & Cuatrecasas P. (1974) Anal. Biochem. 60, 149-152 18 Ikeda S., Sumi Y. & Fukui S. (1974) FEBS Lett. 47, 295-298 19 Campbell J. & Hornby W. (1975) Biochim. Biophys. Acta 403, 79-88 20 Epgasser J.-M. & Coulet P. (1977) Biochim. Biophys. Acta 485, 29-39 21 Coulet P. & Gautheron D. (1980) Biochimie 62, 543 -547 22 Arrio-Dupont M. & Coulet P. (1979) Biochem. D/Biophys. Res. o,.tmmu.n".-". . . . . . . . 0. .~ ., . .D.~ D - - D,~c,, 23 Grasset L., Cordier D. & Ville A. (1977) Biotechnol. Bioeng. 19,611-618 24 Passerat N. & Bolte J. (1987) Tetrahedron Lett. 28, 1277-1280 25 Kurkij~irvi K., Raunio R., Lavi J. & L6vgren T. (1985) in: Bioluminescence and Chemiluminescence: Instruments and Applications (Van Dyke K., ed.), CRC Press, Boca Raton, Florida, vol. 2, pp. 168-183 26 Wilson K., Birchmeier W. & Christen P. (1974) Eur. J. Biochem. 41,471-477 27 Gehring H. (1985) in: Transaminases (Christen P. & Metzler D., eds.), John Wiley, New York, pp. 317-323 28 Soronen R. & Korpela T. (1984) #1: Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos A., ed.), Alan R. Liss, New York, part B, pp. 313-319 29 Harruff R. & Jenkins T. (1976) Arch. Biochem. Biophys. 176,206- 213 30 Harruff R. & Jenkins T. (1978) Arch. Biochem. Biophys. 188, 37-46
439
Immobilization of aspartate aminotransferase on agarose Kalevi KURKIJARVI* and Timo K O R P E L A
Department of Biochemistry, University of Turku, SF-20500 Turku, Finland (Received 11-8-1988, accepted after revision 24-11 - 1988)
S u m m a r y ~ Various methods for immobilization of aspartate aminotransferase (AspAT; from cytosolic fraction of pig heart) on agarose were tested. Aldehyde-, thiol-, and CNBr-activated agaroses were studied in detail. The capacity of the aldehyde support to firmly bind protein was less than 0.2 mg / ml, whereas the apparent remaining specific activity of the bound AspAT was high (50-63% of soluble AspAT). The maximum capacity of SH-agarose to bind enzymatic protein was 3 mg / ml; the apparent remaining activity was 30-40%, and the specific activity determined by Vm~,xwas 51%. Chcr,,ical coupling on to thiol-agarose did not denature the enzyme, as 93% of protein and 83% of the activity were recovered after release of the enzyme from the support. Enzyme protein was quantitatively bound to CNBr-activated agarose (up to 10 m g / m i of the gel). The apparent specific activities were 27-35°/~ while the value calculated from Vmaxwas 46%. Active site-protecting agents within the CNBr-coupling were tested. Bromphenol blue increased the apparent specific activity to 60% and Vm~xto 80% at 3-fold molar concentration at the active sites. Kinetic constants for immobilized preparations were determined. aspartate aminotransferase / agarose / immobilization methods / active site / bromphenol blue / pyridoxal 5'phosphate
Introduction Vitamin B6-dependent enzymes are involved in the central metabolic routes of amino acids and biological amines. A large variety of these enzymes, differing in their catalytic properties, exist in nature. Vitamin B6-enzymes have important applications in large-scale production of amino acids from synthetic precursors [1]. Side effects of PLP-dependent enzymes may also be used in the production of synthetic compounds such as refined chemicals and pharmaceuticals. Recent developments in site-directed mutagenesis for altering specificities with increased capability for producing the enzymes by recombinantDNA methods should give new impetus to the search for practical uses in industry and analytics for this class of enzyme.
In any practical applications of B~-enzymes, the possibility of immobilization on a solid support should be considered because of advantages such as improved stability and reusability [2]. With vitamin-B6 enzymes, this immobilization may be of special importance as many of these enzymes are unstable. On the other hand, their active site contains pyridoxal 5'-phosphate with other catalytic functions that are reactive towards accessible reagent [3], which necessitates proper selection of coupling techniques. This property largely exciudes the coupling reactions that employ activation of the enzyme itself. The present study was conducted to find means of immobilizing PLP enzymes by using cytosolic aspartate amino-transfcrase (AspAT) from pig heart. This enzyme is the most widely studied among the PLP enzymes and details of
*Present address: Wallace Oy, Biochemical Laboratory, P.O. Box 10, 20101, Turku, Finland. Abbreviations: AspAT: Aspartate aminotransferase; EC 2.6.1.1: glutamic-oxaloacetic aminotransferase; PLP: pyridoxal 5'-
phosphate; PMP: pyridoxal 5'-phosphate.
440
K. Kurkijgirvi and T. Korpela
its mechanism and structure have been well documented [3]. Various aspects of the immobilization of AspAT from Escherichia coli on to glass and diatomaceous earth have been recently reviewed by Rozzeli [4].
Materials and methods Chemicals L-Aspartate, L-cysteine, cacodylic acid, malate dehydrogenase (15/~kat / rag), Coomassie brilliant blue G-250, alpha-ketoglutaric acid, pyridoxal 5'-phosphate, and pyromellitic acid were purchased from Sigma Chemical Co., St. Louis, Mo. Phthalic acid, bromphenol blue, and 4-aminobutyraldehyde diethylacetal were from E. Merck A.G., Darmstadt. Glutaric acid, maleic acid, and oxaloacetic acid were from Fluka A.G., Buchs and cyanogen bromide was from Aldrich Chemical Co., Milwaukee W.I. Sepharose CL 4B and activated SH-Sepharose 4B were delivered by Pharmacia Fine Chemicals, Uppsala. The Radiochemical Centre (Amersham, UK) supplied N-ethyl(2,3- C)-maleimide (10mCi / nmol). Bioluminescent NADH Monitoring Kit was kindly donated by Wallac Oy, Turku. The alpha-subform of cytosolic aspartate aminotransferase was purified from pig heart according to Martinez-Carrion et ai. [5]. The specific activity of the AspAT preparations was at least 2.4 p,kat / mg. Immobilization procedures The butyraldehyde derivative of Sepharose CL 4B was synthetized according to Korpela and Hinkkanen [6]. Two g of moist aldehyde-agarose was suspended in 2 ml of 0.1-0.01 M potassium phosphate, pH 7.0-9.0 containing AspAT 0.5- 10.3 mg. The suspension was gently shaken for 20 h at 4oC. The enzyme-gel conjugate was first washed with 50 vol. of 0.01 M potassium phosphate, pH 7.0, containing 1 M KCI and 0.5 mM PLP, followed by 100 vol. of the same buffer without PLP. The azomethine linkages between the gel and enzyme were reduced at 4oC by adding 250/.tl of cyanoborohydride in methanol (1 m g / m l ) after a 20-h incubation of the gel and enzyme in the buffer as above, supplemented with 30% methanol followed by a 30-min incubation with the reducing reagent. The resulting gel was washed with 10 vo! of 30% methanol and 10 vol of water
and finally with 10 vol of 0.1 M potassium phosphate, pH 7.0. The gels were stored at 4°C in 2 ml of the final washing buffer. Activated SH-Sepharose 4B was treated according to the manufacturer's directions. One ml of the swollen gel (0.35 g of the lyophilized powder) was suspended in 2 vol of 0.1 M potassium phosphate, pH 7.0, containing 0.1 M KCi, 1 mM EDTA with AspAT. The suspension was gently shaken at 4°C for 20 h and the gel washed as after CNBr-coupling (see below). The enzyme was released from the support (1 ml) with 50 mM potassium phosphate buffer, pH 8.0, containing 5 mM L-cysteine [7]. Enzymatic activity and protein were measured after overnight dialysis at 4oC against the same buffer containing 0.5 mM PLP. Sepharose CL 4B (2 g of moist, suction-dry gel, equal to 2 ml bed vol) was activated with CNBr by the buffer method (200 mg of CNBr per activation unless otherwise stated) as described elsewhere [8]. The washed activated agarose was normally suspended in 4 ml of 0.2 M potassium phosphate, pH 7.0, containing the enzyme. The suspension was gently shaken at 4oC for 20 h. The resulting AspAT-agarose was first washed with 5-fold vol of 0.01 M potassium phosphate supplemented with 0.5 mM PLP and with 1 mM glycine (pH 7.0), and then with 5-fold vol of 0.01 M potassium phosphate supplemented with 1 M KCi and 0.5 mM PLP (pH 7.0) followed by wash;H. ~. . . . w .i~-h ~ h , a ~ r c t ~ n, H s e c o n d ~a~mch u ~ ~ f l ,,,-~i v~.,, ,.,~z h a t h ing solutions. The suction-dry enzyme-gel was suspended in 2 ml of 0.1 M potassium phosphate, pH 7.0, containing 0.5 M KCI and stored at 4oC. ,,JlU?
I~IFJI+ l , . J l k . , t t r L l l
Lll'Ik,,,
III~II.
~&
I'I~
TvU~-I+I--
Determination of protein and enzymatic activity Soluble proteins were measured with the biuret assay against bovine serum albumin, and the Coomassie brilliant blue [9] method (for dilute solutions) was standardized against it. Sepharose-bound protein was calculated from the differences between the soluble protein before and after the coupling process. Except for the studies carried out with SH-Sepharose, proteins were also determined by N[t4C]-ethylmaleimide-labeled AspAT added to the enzyme to be coupled [ 10]. These 2 methods correlated well; the radioactive method usually showed only a few percent less bound protein concentrations. The AspAT enzymatic reaction was measured kinetically from alpha-ketoglutarate and Laspartate (both 20 mM unless otherwise mentioned to oxaloacetate and glutamate at 25°C in 0.1 M potassium phosphate, pH 8.0. Enzyme activ-
Immobilized aspartate ami~,u;~rat!,:ferase ity was expressed as micromoles of oxaloacetate formed per second (v&at). Soluble enzyme was normally measured at a protein concentration of 1 g g / m l . The immobilized enzyme was measured with a stirred batch reactor [11, 12] using 20 mg of suction-dry enzyme-gel in the reaction vol of 13 ml. Enzyme-bound PLP was measured fluorometrically after release of the coenzyme with 5.5% ( w / v ) trichioroacetic acid at 50°C. Bioluminescent determination of the active centers and maximum rate calculations are presented elsewhere [ 12].
Results
Butvraldeh y d e - agarose Cy(osolic pig heart aspartate aminotransferase contains 19 lysine residues per subunit [13], part of which could react with aldehyde groups on solid support through the Schiff base reaction under mild chemical conditions. AspAT retained its activity very well, buc coupling efficiency was low (Table I). The latter result was obviously due to the fact that the Schiff base linkages were formed reversibly and the enzyme therefore leaked out of the gel during extensive washing. Coupling efficiency increased at low iotfic strength (10 raM) and at higher pH (9.0) of the reaction and washing buffers, but AspAT was uetacucu immeuiately when higher salt was used or the pH was lowered. Ion-exchange interactions were probably responsible for binding of this elutable protein. Although the percent coupling was low (Table I), it could be seen that a small part of the enzyme was stably bound and was not lost during extensive use under reactor conditions. High apparent (observed) specific
441
activity was due, in part, to the low bound enzyme activity resulting in low diffusional limitations. Fixation of the imino bonds by reduction with cyanoborohydride ;~ a routinely used method for stabilizing them [14]. !n ,the case of PLP-enzymes the reduction may noz be favorable because of the subtle reduction in the PLP-apoenzyrne Schiff base linkage with concomitant loss of catalytic activity [3, 4]. In a test in which the Schiff base linkages were reduced, the coupling efficiency increased 10-fold with complete loss of activity. This confirmed the presence of a substantial amount of sterically available reactive aldehyde groups on the gel. Immobilization c f A s p A T on cvanuric chloride- and divinyl-sulfone-activated agaroses [15] was tested. In both cases only 3 - 5 % of activity was found after immobilizati,3n. The low figures can be explained by the poor elutability of the activating compounds from the gels.
Immobilization on thiol-agarose AspAT contains 5 SH-functions per monomeric enzyme molecule, 3 of which can be modified with soluble reagents, whereas the final 2 cysteinyl residues react only under extensive denaturing conditions [16]. Immobilization on SHfunctions is of special interest since the enzyme can be detached with soluble thiols and the resulting enzyme comp:~rocl u, ith th~ ,~,4,,,;,,~I ,,,a,~,identical reaction conditions. Coupling of AspAT to SH-agarose was incomplete at any concentration, while maximal binding capacity was ~3 m g / m l of gel (Fig. 1)o Apparent bound activity was found to be linearly dependent on the bound protein up to =1.5 m g / m l of gel with an enzyme preparation with asp. act. of 3/~kat/rag. The apparent spe--
.
_ .
.
.
.
~
.~
t v.l.t
ttt~
1o, lt I~III~LI
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Table I. Immobilization of AspAT on butyraidehyde-agarose. Protein (rag)
Activity (#kat)
Coupled protein (mg/ml)
Coupled activity (/.tkat / ml)
Apparent S.P. Act. (/xkat / mg)
I).5 1.0 2.6 5.1 10.3
1.2 2.4 6.3 1'2.3 24,9
(1.02 0.04
0.03 0.05 0.09 0.15 0.25
1.50 (63) 1.25 (52) 1.29 (54) 1.50 (63) 1.19 (50)
0.07
0.10 0.21
AspAT was immobilized without cyanoborohydride reduction as described in Materials and methods. The specific activity of the AspAT preparation was 2.4 ttkat/mg. The proteins were measured by radioactively labeled enzyme. The percentage specific activitiescompared to the solu01eenzyme are shown in parentheses.
K. Kurkijiirvi and T. Korpela
442
-z""0
of enzyme activity during the procedures were taken into account practically all of it was recovered in the soluble form. No significant differences in the immobilization properties of the gel were observed when immobilization was repeated 3 times. The enzyme itself did not denature upon immobilization (see Fig. 3 and above). One obvious explanation for the apparent loss of activity is the mass transfer barriers provided by the
I
.",b
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0.50
u
Ij
i
..
A~,pAT ;t4 ZtlF.!OE::'L : "'r~"~r' . . . .r.- ,.-._., r.r., - E an!._';~
' . ' . h_ ;q-r:L)
Fig. 1. Capacity of S H - S e p h a r o s e to bind AspAT. The solution of immobilization contained 3 ml of 0.1 M potassium phosphate, pH 7.0, 0.1 M KCI, 1 mM EDTA, 1 ml of t h e ool
anti 1] 3 - - 0 ~ . . . . . . . . . . . . . .
mo
nf
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nrl~tPin i~ .......
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3.0 /zkat/mg). The reaction was allowed to proceed for 20 h at 4°C. (a) bound protein; (b) apparent bound enzyme
" -'I--
.
I0
15
.:ri
25
IHHOB]LZZFITIOH TIHE ,:hours:,
Fig. 2. Binding kinetics of AspAT on SH-Sepharosc. The immobilization took place in 24 mi of potassium pht)sl~hatc, pH 7 0 containing 0.1 M KCI with 8 ml of moist gel and 24 mg of AspAT (specific activity 3.(1/xkat/mg) at 4"C. Samples were assayed from the su,,;pension at appropriate time intervals. The results are expressed percentually of the soluble enzyme at the beginning of the procedure in the immobilization solution when bound protein and activity is 0 % and the supernztant protein and activity is 100%.
activity.
% OF :[[41TI,.:iL qL':TIVIT"/
I00
cific activity was 3 0 - 4 0 % of the soluble one in the valid range of enzyme concentrations. The time-course of coupling of AspAt on SH-agarose (Fig. 2) was similar to the reaction rate of soluble reagents [16]. A tendency to 3 different reaction steps was found (Fig. 2). Protein binding and activity correlated well, suggesting that the reaction product was equal with respect to activity remaining within the advance of the reaction. A preparation of immobilized AspAt containing 2.8 mg protein / ml of the 8c! was eluted from the gel with 5 mM cysteine [7]. After dialysis against buffer containing 0.5 mM PLP, the recovered percent ot protein was 93 while that of enzyme activity was 83. The immobilized gel did not show any activity after cysteine washing with PLP-containing buffer. When normal losses
.
~
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.
.111 I
~
k
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, - , F'LP ;'ERCr ] ..,.
i
50
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.,,
"." ~ F'IIF' r,JE,, 25 •
hi. ' m ,
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20
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7n
40
50
e.U
,-,t i
J'~,_d=,HTIur~ rIHE (r~,jn;,
Fig. 3. Thermal stability of SH-Sepharose-immobilizcd AspAT. The immobilized preparation ( I mg AspAT / ml of gel) was incubated at 70,'C in 20 mM potassium phosphate, pl! 8.0 (15 mg of moist gel in 2.5 ml of the buffer) containing eithel ! mM L-aspartate or 1 mM alpha-ketoglutarate. The enzyme reactivated in the presence of i,-aspartate (PMPform) was reactivated by incubating the enzyme in 20 mM potassium phosphate, pH 8.0, containing 10 mM PLP at 5"C with gentle shaking.
Immobilized asparlate aminotransferase gel network. They can be minimized by reciprocal measuring of the apparent activities at high substrate concentrations [12]. In this manner Vm~xof 51% of the soluble enzyme was achieved (Table II). Since the Vmax values were nearly constant and the Km values reasonable compared with the soluble enzyme, at least up to ~1 rag, enzyme p r o t e i n / m l of gel (Table II), the Vm~ value for bound A s p A T (51%) was reliable. Hence, the 49% reduction in enzyme activity most probably resulted from modification of thiol groups on AspAT, While the half-life of the enzyme activity of A s p A T - S H - a g a r o s e at 4°C in 0.1 M potassium phosphate, pH 7.0 supplemented with 1 M KCI was ~ 2 y, that of soluble enzyme was 2 mo. Good storage stability, with distinctly increased thermostability of the immobilized A s p A T (r~/2 = 10 h at 70oC) compared with soluble AspAT in the PLP form (rl/2 = 2 h), suggested that at least one covalent bond to each monomeric unit existed. When the enzyme was transformed to the PMP form with aspartate [12], the half-life of inactivation at 70"C was ~5 min (Fig. 3), which was exactly the same as with the soluble enzyme; i.e., immobilization did not protect ,L~,,,,:PMP form of the enzyme. The enzyme was, bowever almost completely reactivatable flora the stage of 50% inactivation with PLP treatment (Fig. 3), whereas in the absence of PLP no reactivation appeared.
443
CNBr-activated Sepharose Binding capacity and binding strength in immobilization can be controlled both by varying the activation degree of the gel and by the pH of the immobilization reaction. Both these factors depend on the properties of the enzyme and on the pH-reactivity profile of the support. The immobilization reaction was carried out at pH 6.0, 7.0, and 8.0, at different degrees of activation. Figure 4 shows that protein binding capacity was about the same at pH 7.0 and pH 8.0, but the observed activity of the preparation bound at pH 7.0 was almost ?-,Cold higher than at pH 8.0. Immobilization at pH 6.0 showed lower protein coupling and remaining activity than at neutral pH (Fig. 4). The apparent optimum for CNBr concentration in the activation was ~50 m g / ml of the gel (Fig. 4) with the remaining activity used as the criterion. HoweveL the result was no longer optimal for thermostability of the preparation (Fig. 5). For further experiments, activation with 100 mg C N B r / m i of agarose was chosen as a compromise. The capacity of the CNBr-activated agarose to bind A s p A T was tested by varying the amount of enzyme in the immobilization. Bound protein increased in a strictly linear fashion as a function of the enzyme, and no saturation concentration was reached up to 9 m g / m l (Fig. 6a). On the contrary, an apparent saturation of bound
Table il. Comparison of the Km values for r-aspartate (KmA) and for alpha-ketoglutarate (KrnB) and the Vmax
velocities for immobilized and soluble AspAT. A Km (mM)
B Km (mM)
Vmax (p,kat / mg)
Soluble AspAT
4.(I
0.8
3.5
AspAT-Stt-agarose 0.30 mg / ml 0.60 mg / ml 1.00 rag/ml 2.81) mg / ml
4.4 4.9 5.4 6.5
1.7 1.9 2.6 5.0
1.8 1.8 1.7 1.4
AspAT- CN Br-aragose 0.35 m g / m l 1.30 mg/ml 2.70 rag/ml 4.00 mg/ml
2.5 3.3 4.2 4.1
1.5 1.9 2.4 2.4
1.6 1.7 1.4 1.2
Enzyme preparation
The concentration of soluble AspAT (specific activity 2.4-3.11/~kat/mg) was 1 /~g,/ml and immobilized enzyme was used 11)-25 mg of moist gel in the reaction volume of 13 ml [12].
K. Kurki#irvi and T. Korpela 4 SC~J,'4DFP.OTEIH c.rL=.:, a mlm~UlI~UlUlUIIIIIIIIIII
i ¢# i
,.,pH 8.0 g
• --pH 7.0 I #
pH 6.0
0
5o
10o
z,:,_-,
ISo
CHBr'GEL (fi,g'hlL)
BOUNDQCTIV!TY(ukavmL) •
~
i
i.o
iiliiIIIIIIIIIIIIiiiI l
0.5
,$
' '
m,mt
,-,pH 8.0
--
• .-F,H 7.0
--~
--pH 6.0
0.0
0
5o
i0o
r
. . . .
6.0 4.0
Fig. 4. The effect of the number of reactive groups on CNBractivated Sepharose on AspAT immobilization at different pHs. With each immobilization, 3.6 mg of AspAT (specific activity 2.4 t~kat / mg) were employed. Immobilization was carried out in 0 2 M n• n t a K ~ i H m nhncnhat~ hi--I[ K f l '7 f l n r ~ i~=. v.v~ , .v~ vl 8.0. (a) boun-d protein; (b) bound apparent enzyme activity. . . . . . . . . . . .
BOUND PROTEIH (mg,r~,L,:,*,_=el:."
8.0
2oo
15o
CNBr/GEL (mg4E;,
. . . . . .
enzyme activity was reached at about 3 m g / m l (Fig. 6b). Immobilization of proteins has been reported to occur on CNBr-activated agarose within 2 - 3 h at room temperature and within = 2 0 h at 4°C [17]. Kinetics of immobilization on to CNBractivated agarose was studied at 4°C by measuring the specific activities of the enzyme in the coupling buffer and in the gel-bound stage (Fig. 7). More than 90% of protein was bound in 6 - 8 h; however, no increase in the apparent enzyme activity was found after 2 - 3 h (Fig. 7). A special feature of the experiment shown in Fig. 7 was that enzyme activity decreased faster than the bound activity increased. This finding prompted us to study further the effect of the washing time of the gel, since it was conceivable that the enzyme inhibition was caused by free CNBr in the solution. It has been recommended
I~
2.0 PROTEIN
0.0
o
ni.=,
i
. . . . .
AzpAT !!-IINNOE',ILIZATIOHBUFFER c:rYLl.'r~,L) BOUND ACTIVITY (ukat/mL)
% OF IH'rTIRL ACTIVITY 100
-,~ .~
. ....~.._..
2,5"
_ -
CNBr 20n n,9
.
• ,..,.......
75
,...
50
on
C.I-
"- -.
I00 ~g --...
1.5
...
.-...,.
1.0
-.
50 ~9 -..
0.5 25 ~9 SOLUBLE
ACTTVITY 0.0
•
u 0
~
2
3
,7.5
i
1.5
4 AspAT IN INMOBILISATION BUFFER O,,g..'~,L)
TINE (hours)
Fig. 5. Thermal stabilities of AspAT's immobilized at different CNBr-activation degrees. The conditions and preparation of the experiments were as in Fig. 3. The soluble enzyme was treated in the same conditions at concentration of 0.15 mg/ml.
Fig. 6. Binding of AspAT on CNBr-activated Sepharose. Immobilization was carried out at 4°C (20 h) in 6.0 ml of 0.2 M potassium phosphate, pH 7.0, containing 2 ml of moist AspAT-agarose carrying 0.3-9.0 mg of enzyme (specific activity 2.4 /xkat/mg). (a) bound protein; (b) apparent bound enzyme activity.
Immobilized aspartate aminotransferase in the general procedures for CNBr activation that washing and transfer to the coupling solution be done within <2 min to obtain maximum coupling efficiency [17]. We found that when washing time was increased from 1.5 min (1.5 m l / s to 5 - 7 min, the observed activity of the immobilized AspAT was 10% U higher than with the shorter washing time, while the coupling efficiency of the protein remained the same. Washing for <1.5 rain clearly showed that either unreacted CNBr or another active compound released from the activated gel was responsible for the enzyme inhibition. The latter
445
alternative was also implied by the prolonged decrease in the specific activity of the soluble enzyme (Fig. 7, compare with Fig. 2). The apparent specific activities of immobilized AspAT preparations were from 27% to 35% in the region of valid enzyme ioadings extending up to ~ 1.5 m g / m l of gel. According to the measured V~ax values (Table II), the specific activity yield of 46% was obtained as calculated from the smallest protein loading. As with SH-Sepharose, the Km values were related to the soluble enzyme with slightly lowered Km for aspartate and increased for 2-ketoglutarate.
Protection of AspA T against activity losses OF USED I00 i I,AHt i i~ I~ i I H II # nl 0 l,~ i b ill i , I lm I ::
"
75 6"
#
.50
, - , BOUHD ACTIVITY ---BOUND PROTEIH SP. ACTIV.
f f
/1~
i ill i JlI l l l l i l
I l l l I l I l I I I I l I I
25
soluble 0
-
4
6
R
IHt4OBILIZATIOH TItlE (hours)
Fig, 7, Binding kinetics of AspAT on CNBr-activated agarose. Immobilization took place at 4°C in 22 m! of 0.2 M potassium phosphate, pH 7.0, containing 11.5 mg of AspAT (specific activity, 2.4/.tkat/mg). The results are expressed in percent of the soluble enzyme at the beginning of the coupling procedure (see text in Fig. 2).
Since AspAT seemed to be sensitive to inactivation by soluble reagents during the immobilization procedures, potentially protective compounds in the coupling buffer were studied (Table III). The coenzyme PLP did not show any effect on immobilization results, indicating that coenzyme loss did not occur during immobilization. A slightly lower activity yield was actually observed with glutarate and maleate. In the presence of more bulky aromatic carboxylic acids, pyromellitic acid and phthalic acid, a reduction in activity was also obtained. Specific activity was slightly improved with slightly lower binding of the protein in the presence of both substrates. Thi,s may, however, be an artefact because o'~ reacts to the gel through its amino aspart,~,~, group and thus the result reflects the effects of a less reactive gel. Cacodylate and bromphenol blue significantly
Table I i l , Effect of active site-protecting c o m p o u n d s on immobilization of A s p A T on CNBr-activated agarose.
Additive
Bound protein (rag/ml) (%)
Bound sp. activity (~kat / m,~) (%)
None
1.5
(94)
(I.65
(27)
10 mM glutarate 10 mM maleate 10 mM pyromellitinic acid l0 mM phthalate l0 mM L-aspartate-ketoglutarate 0.5 mM PEP 0.01% Bromphenolblue ' 0.2 M cacodylate
1.5 1.4 1.4 1.4 1.3 1.2 1.4 0.6
(94) (88) (88) (88) (81) (75) (88) (38)
0.45 0.38 0.58 0.27 0.85 0.65 1.44 1.22
(19) (16) (24) (1 l) (35) (27) (60) (51)
The specific activity of soluble AspAT was 2.4/.tkat / mg which was used at a concentration of 1.6 m g / ml of gel. The immobilization was performed in 0.2 M potassium phosphate (0.2 M, pH 7.0), except in the case of potassium cacodylate which replaced the potassium phosphate (pH 7.0). The specific activities were measured in standard conditions involving 20 mM substrates. The percentage coupling efficiences are presented in parentheses. Bound protein was determined with radioactively labeled AspAT.
446
K. Kurkijiirvi and T. Korpela
increased the immobilized specific activity (Table III). Of the two, bromphenol blue was more favorable because it did not affect the coupled protein. Maximal protection by bromphenol blue was obtained at 0.01% ( w / v ) concentration (Fig. 8). Table IV shows Vm~, values for AspAT with measured PLP content, transaminable active sites, and turnover rate for the soluble enzyme that immobilized conventionally and that which immobilized in the presence of bromphenoi blue. The Vm~,xof the preparation immobilized in the presence of bromphenol blue was almost twice that in its absence. The coenzyme was not significantly lost in either case. The number of active transamination centers drastically increased in the presence of bromphenol blue, while their average turnover increased only slightly (Table IV). Because added PLP did not shield the enzyme from activity loss (Table III) and because the measurable PLP content with and without bromphenol blue was practically identical (Table IV), the free CNBr reacted to the active site to a function other than PLP. This was also supported by immobilization of the PMP form of AspAT which showed the same remaining activity as the PLP form procedure.
Discussion In the 1970s a number of studies were published on AspAT immobilization, lkeda et ai. [18] showed that the apoenzyme is bound similarly to the holoenzyme on CNBr-activated agarose, indicating that iysine-258 is not reactive. Immobilized AspAt was used to measure L-aspartate in micromolar quantities [18]. In addition, AspAT was immobilized on aminoethyl cellulose [19] and on collagen film [10, 20, 21] and immobilized
BOUNDSP. RC~IVITY (ukatx~g)
1,5
1,0
0.5 0
0.:]04
0.008
0.012
BROMPHEHOLBLUE (%P Fig. 8. Effect of the concentration of bromphenol blue on AspAF immobilization on CNBr-activated Sepharose. The conditions of the experiments are similar to those in Table II1.
AspAT was used as a kinetic model of 2-substrate enzyme reaction. Arrio-Dupont and Coulet (1979) studied AspAT subunit interactions by using collagen film-immobilized enzyme and showed that only the dimeric enzyme is active [22]. Woven silk was used as the support for immobilized AspAT by Grasset et al. [23]. In the 1980s, events taking place during the immobilization of AspAT on CNBr-activated agarose were studied in detail [12], and later, immobilized AspAT was used coupled to bioluminescence enzymes to measure L-aspartate and oxaloacetic acid at the picomole level [25]. Recently interest in the immobilization of AspAt seems to have increased with promising results en the utilization of side effects of AspAT for production of certain metabolites [4, 24]. Although immobilization efficiency on aldehydic agarose containing 4-carbon spacer was
Table IV. C o m p a r i s o n of properties of soluble A s p A T with c o n v e n t i o n a l l y immobilized A s p A T 1 and A s p A T
immobilized in the presence of 0.01% (w/v) Bromphenol blue (AspAT 2). Parameter
Soluble
Bound AspAT 1
Bound AspAT 2
Vm~,x(p.kat / rag) PEP (nmol / rag) Transaminationable active centers (nmol / mg) Turnover number (~kat / nmol)
3.08 12.8
1.26 (41) 11.0 (86)
2.46 (80) 11.5 (90)
12.8 0.24
6.7 (52) 0.19 (79)
11.0 (86) 0.22 (93)
The .percentage values compared to the soluble enzyme are shown in parentheses. The turnover numbers were calculated as the maximum velocity per "'transaminationable" active center.
hnmobili: od aspartate aminotrans]erase low, this method yielded the highest operative specific activities (Table I). Glutardialdehyde coupling and dialdehyde supports achieved by periodate oxidation have often been used for immobilization of enzymes with borohydride reduction [15]. These reactions are complex and also involve reactions other than the Schiff base reaction. The method employed here was more straightforward and novel in ~he context of protein immobilization and the results show that a small portion of the enzyme was firmly bound to the gel, although the Schiff base linkage, as such, may not be stable enough. Thus, the mechanism of binding must involve a multipoint attachment of the enzyme at specific microenvironments on the gel. A large number of aldehyde functions remained free as proved by a 10-fold increase in bound protein in the presence of cyanoborohydride. The aldehyde matrix employed could also serve as a special chromatographic material capable of separating proteins and other substances according to the number of amino functions. S H - a g a r o s e can be considered as a modifying reagent; compared with the soluble reagents it has steric hindrances which may be utilized in studies of the SH groups located on the surfaces of proteins. Cysteines 45 and 82 of cytosolic AspAT react readily with soluble thiol reagents. Within their modification, activity varies between 5 6 - 1 2 5 % of that of the original enzyme [26]. The third modifiable residue, cysteine .~,v, ~"~" r e a c t s , syncataiyticaiiy, in the presence of substrates with an activity decrease to < 5 % , reflecting conformational movements necessary for catalysis [16, 26, 27]. Since the remaining activity of the support-bound enzyme was ~50% and since the enzyme was immobilized in the absence of substrates, cvsteine 3911 scarcely took part in the process. An3, slow reaction to cystein-390 did not occur because the preparation was stable over extended periods. Against the background of the cited modification studies by soluble reagents, the present loss of enzyme activity upon immobilization most probably resulted from decrease of flexibility of the enzyme molecule when bound through cysteinyl residues 45 a n d / o r 82. Immobilization on SH-agarose did not protect the PMP form of AspAT from denaturation as compared with the soluble enzyme. Fig. 3 shows that denaturation commences with a reversible release of PMP followed by more or less reversible denaturative stages of the apoprotein. Because the enzyme was bound through at least
447
2 covalent bonds to the solid matrix, dissociation into subunits was not involved in the procedure. Conversely, the results suggest that denaturation of soluble enzyme follows the same mechanism. The lack of protection of the PMP form of AspAT by immobilization also supports the argument that the enzyme was bound to the support through SH functions located relatively far from the active sites. Chromatographic behavior of heat-treated AspAT has shown that while enzyme activity remains unaltered, irreversible changes in the apoprotein can occur in the enzyme [28]. Hence denaturation of the active site region and of other parts can occur independently. A remarkable property of immobilization on CNBr-activated agarose was that the remaining activity was very sensitive to the washing time of the activated gel. The present results show that successful immobilization of vitamin B6-dependent enzymes requires extraordinary care when gel-activating reagents are removed. With other enzymes, traces of the reagents may not impair the yields, but with B6 enzymes this is not the case. Hypotheses about free CNBr and another solubilized reagent (e.g., trimerized CNBr) in the ceupling buffer led us to study active siteprotecting compounds. The most significant finding was that bromphenol blue dye was a very effective agent, increasing the remaining activity ~2-fold compared with conventional coupling with moderate whashing time of the gel (Table IV). Bromphenol blue was effective in concentrations of about 3-fold the molarity of enzyme. When one considers probable binding of the dye on the agarose matrix, binding to the enzyme was nearly quantitative. The shielding was obviously due to the !act that bromphenol blue binds reversibly at or near to the active site with Kl of 0.6 x 10- ~'[291. Other mono- and dianions, including cacodylate, also bind pH-dependently to AspAT [30]. The dicarboxylate acids tested (Table III) decreasfd the activity rather than increasing it, which was somewhat'surprising for they are known to induce a - closed conformatioil ,, of the active site [3] and it was conceivable that this would prevent reactions near the active site. Because bromphenol blue and cacodylate are both anionic compounds at pH 7.t), they may protect by binding to the same positively charged function and thus only one catalytically important function may be the subject of protecflop. These observations show that active-site protecting compounds can be found among known reversible inhibitors of the enzyme.
448
K. Kurkijiirvi and T. Korpela
The calculated average turnover rates and the K m values (Table II) showed that the properties of the enzyme changed only slightly upon immobilization, suggesting that the active sites were not embedded in gel, thus the chemical milieu was not essentially different from the soluble reaction. While bromphenol blue clearly protected against soluble reagents, it also slightly increased the average turnover rate (Table IV), which shows that it shielded the enzyme from modification by the solid reagent. The active site-protecting agents of the present study were quite different from other studies with AspAT from E. coli [4]. Our attempts to employ carbodiimide reaction of coupling yielded inactive enzyme, whereas E. coli enzyme has been successfully immobilized by carbodiimide in the presence of substrates and PLP [4]. On the other hand while A s p A T retained its activity when immobilized on S H - a g a r o s e well, alanine aminotransferase completely lost activity in the process (unpublished observations). These aspects show that enzymes catalyzing similar reactions can behave quite differently upon immobili~:ation, as is the case with modifications by soluble reagents. As stated by Rozzell [4], a common feature of transaminases and other vitamin B6-dependent enzymes seems to be dependence on suitable immobilization chemistry.
References 1 Soda K., Tanizawa K., Esaki N. & Tanaka H. (1987) in: Biochemistry of Vitamin B6; Proceedings of the 7th International Congress on Chemical and Biological Aspects of Vitamin B6 Catalysis (Korpela T. & Christen P., eds.), Birkh/iuser Verlag, Basel, Boston, pp. 435-444 2 Lilly M. & Dunnill P. (1976) in: Methodsin Enzymology (Mosbach K., ed.), Academic Press, London, vol. XLIV, pp. 717-738 3 Arnone A., Christen P., Jansonius J. & Metzler D. (1985) in: Transaminases (Christen P. & Metzler D., eds.), John Wiley, New York, pp. 326-362 4 Rozzell J. (1987) in: Methods in Enzymology (Mosbach K., ed.), Academic Press, London, vol. 136, pp. 479-497 5 Martinez-Carrion M., Turano C., Chiancone E., Bossa F., Giartosio A., Riva F. & Fasella P. (1967) J. Biol. Chem. 242, 2297-2409 6 Korpela T. & Hinkkanen A. (1976) Anal. Bio-
chem. 71,322-323 7 Carlson J., Axen R., Brocklehurst K. & Crook E. (1974) Eur. J. Biochem. 44, 189-194 8 Korpela T. & Kurkij/irvi K. (1980) Anal. Biochem. 104, 150-152 9 Bradford M. (1976) Anal. Biochem. 72,248-254 10 Coulet P., Godinot C. & Gautheron D. (1975) Biochem. Biophys. Acta 391,272-281 11 Kurkijarvi K. & Korpela T. (1981) Biotechnol. Bioeng. 23, 1389-1392 12 Kurkij~irvi K. & Korpela T. (1983)Appl. Biochem. Biotechnol. 8, 135-144 13 Barra D., Bossa F., Doonan S., Fahmy H., Hughes G., Martini F., Petruzzelli R. & Wittman-Liebold B. (1980) Eur. J. Biochern. 108, 405 -414 !4 Parikh I., March S. & Cuatrecasas P. (1974) in: Methods in Enzymology (Jakoby W. & Wilchek M., eds.), Academic Press, London, vol. 34, pp. 77-102 15 Turkova J. (1978") in: Affinity Chromatography. Elsevier, Amsterdam, pp. 153-189 16 Birchmeier W., Wilson K. & Christen P. (1973) J. Biol. Chem. 248, 1751-1759 17 March S., Parikh I. & Cuatrecasas P. (1974) Anal. Biochem. 60, 149-152 18 Ikeda S., Sumi Y. & Fukui S. (1974) FEBS Lett. 47, 295-298 19 Campbell J. & Hornby W. (1975) Biochim. Biophys. Acta 403, 79-88 20 Epgasser J.-M. & Coulet P. (1977) Biochim. Biophys. Acta 485, 29-39 21 Coulet P. & Gautheron D. (1980) Biochimie 62, 543 -547 22 Arrio-Dupont M. & Coulet P. (1979) Biochem. D/Biophys. Res. o,.tmmu.n".-". . . . . . . . 0. .~ ., . .D.~ D - - D,~c,, 23 Grasset L., Cordier D. & Ville A. (1977) Biotechnol. Bioeng. 19,611-618 24 Passerat N. & Bolte J. (1987) Tetrahedron Lett. 28, 1277-1280 25 Kurkij~irvi K., Raunio R., Lavi J. & L6vgren T. (1985) in: Bioluminescence and Chemiluminescence: Instruments and Applications (Van Dyke K., ed.), CRC Press, Boca Raton, Florida, vol. 2, pp. 168-183 26 Wilson K., Birchmeier W. & Christen P. (1974) Eur. J. Biochem. 41,471-477 27 Gehring H. (1985) in: Transaminases (Christen P. & Metzler D., eds.), John Wiley, New York, pp. 317-323 28 Soronen R. & Korpela T. (1984) #1: Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos A., ed.), Alan R. Liss, New York, part B, pp. 313-319 29 Harruff R. & Jenkins T. (1976) Arch. Biochem. Biophys. 176,206- 213 30 Harruff R. & Jenkins T. (1978) Arch. Biochem. Biophys. 188, 37-46