The Use of Diazenedicarboxylic Acid Derivatives for Protein Cross-Linking : Use of Diazene for S-S Cross-Linking of Aldolase

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  • Eur. J. Biochem. 100, 393-398 (1979)

    The Use of Diazenedicarboxylic Acid Derivatives for Protein Cross-Linking Use of Diazene for S-S Cross-Linking of Aldolase

    Maria T. MAS, Janina BUCZYLKO, and Marian KOCHMAN

    Department of Biochemistry, Technical University of Wroclaw

    (Received May 16, 1979)

    1. The reaction of the diazenes 1,2-diazenedicarboxylic bis(N-methylpiperazide) (I), 1,2-di- azenedicarboxylic bis(NJVdimethy1piperazide) diiodide (11) and 1,2-diazenedicarboxylic bis(N-me- thylamide) (111) with aldolase was studied. The reactivity of the diazenes with free -SH groups decreased as follows: 11 > I > 111. At pH 6.5 and 5 C, moderately slow disulfide bond formation occurs in protein due to interaction with diazene I. The mechanism of the reaction proceeds by formation of an intermediate, [RNCONHN(S-aldolase)CONR]. This intermediate can be separated from the reaction mixture and then used for -S-S- cross-linking with - SH-group-containing mole- cules. In the absence of free - SH groups a slow decomposition of the diazene-aldolase intermediate proceeds within several days.

    2. It has been demonstrated that this diazene-aldolase intermediate as well as the corresponding derivative of thiopropyl-Sepharose 6B can be used for immobilization of the enzyme to the solid matrix via -S-S- bonds.

    3. The oxidation of aldolase -SH groups induced by diazene I results in the formation of soluble aldolase polymers. The unimer ( M , 160000), dimer, trimer and higher molecular weight polymers retain an average of 50 % of the initial enzymatic activity. Comparison of kinetic properties of aldolase polymers and native aldolase showed that cross-linking slightly influenced the catalytic properties of the enzyme. After reduction with dithiothreitol, aldolase polymers form unimers with a specific activity equal to that of native aldolase.

    Recently, diazenedicarboxylic acid bisamides have been described as thiol-oxidizing agents with high specificity toward glutathione. The mechanism of glutathione oxidation has been studied in detail by Kosower and coworkers [2]. It was proposed that the first step involves addition of thiolate to the N = N bond with formation of a rather unstable intermediate [Eqn (111:

    Abbreviations. Diazene I, 1,2-diazenedicarboxylic bis(N-me- thylpiperazide); diazene 11, 1,2-diazenedicarboxylic bis(N,N-di- methylpiperazide) diiodide; diazene 111, 1,2-diazenedicarboxylic bis(N-methylamide); Nbsz, 5,5-dithio-bis(2-nitrobenzoic acid); Mops, 3-(N-morpholino)propane sulfonic acid.

    Enzyme. Aldolase (EC Note. The term unimer has recently been proposed for use in

    discussing association-dissociation phenomena in macromolecular chemistry [l]. We use this term with respect to the aldolase molecule ( M , 160000) composed of four subunits. Dimer, trimer and higher polymers are referred to as the associated unimers of aldolase of molecular weight 320000, 480000 and higher molecular weight, respectively.

    R-SH + R-C-N = N-C-R -+ R-C-N-N-C-R (1) I I

    I H+ R

    S H


    COR HH

    RCONNHCOR+ H20-+RrCO%NHCOR f R-SdH2 I S (3) I R

    R-SdH2 -+ R-SOH + H + (4)

    ( 5 )


    0 I/

    R-SOH + R-SOH -+ R-S-S-R + H20 R-S-S-R + H20 -+ R-SH + R-S-OH

    II II 0 0

  • 394 Use of Diazene for Cross-Linking of Aldolase

    where R represents a residue of a thiol compound and R' = N(CH2)4NCH3 with the N(CH2)4N in a piperazine ring. The second step of the reaction involves disulfide bridge formation with free -SH groups of the thiol compound [Eqn(2)]. In the absence of free sulfhydryl groups, decomposition of the intermediate occurs according to Eqn (3). This re- action may initiate a recovery of free thiolate group or may lead in several subsequent steps to partial oxidation of sulfhydryl groups to -S02H derivatives

    Diamide-induced formation of rat hemoglobin- glutathione mixed disulfides has been demonstrated [3,4]. According to Harris and Biaglow [ 5 ] and Koso- wer et al. [6] 1,2-diazenedicarboxylic bis(methy1- amide) reacts poorly with -SH groups of bovine serum albumin, coenzyme A and human hemoglobin. Although diazene derivatives oxidize some thiol groups in proteins [7], very little effect on the activity of some glycolytic enzymes [8] and no effect on papain activity has been observed [S].

    In our laboratory a spontaneous aggregation of rabbit muscle aldolase with accompanying 50 loss of activity was observed after 1 - 3-years storage of crystalline enzyme suspension in ammonium sulfate solution at pH 7.5. It was found that this process was accompanied by intermolecular disulfide bond for- mation. Aldolase polymeric forms were converted into monomers by reducing agents. The aim of this study is to investigate the possibility of utilization of diazenedicarboxylic bisamides for intermolecular cross-linking of aldolase, to analyse the reaction course and characterize the molecular and catalytic pro- perties of the enzyme during the - SH group oxidation, The possibility of use of diazenes for immobilization of enzymes has also been investigated.

    [Eqn (4 - 6)1 PI.


    Reagents and Enzymes

    Fructose 1,6-bisphosphate, NADH, 5,5'-dithio- bis(2-nitrobenzoic acid) (Nbsz), dithiothreitol, triose- phosphate isomerase and glycerol phosphate dehydro- genase were purchased from Sigma Chemical Co., Tris from Mallinrodt, urea (ultra pure) from Schwartz/Mann, sodium dodecyl sulfate and NaBH4 from BDH Chemicals Ltd, 3-(N-morpholino)propane sulfonic acid (Mops) and its sodium salt from Cal- biochem. Sephadex G-200, Sephadex G-25 and thio- propyl-Sepharose 6B were obtained from Pharmacia Fine Chemicals, All other chemicals were purchased from POCh Gliwice.

    Diazenedicarboxylic acid bisamides : 1,2-diazene- dicarboxylic bis(N-methylpiperazide) (I) 1,2-diazene- dicarboxylic acid bis(N,N'-dimethylpiperazide) diio-

    dide (11) and 1,2-diazenedicarboxylic bis(N-methyl- amide) (111) were synthesized by Dr L. Syper (In- stitute of Organic and Physical Chemistry, Technical University of Wroclaw) according to Kosower [2]. All reagents were of analytical reagent grade. All solutions were prepared in deionized water.

    Rabbit muscle aldolase was prepared according to Penhoet et al. [9]. Specific activities of aldolase preparations were in the range 16- 19 units/mg protein. Aldolase activity was assayed spectrophoto- metrically in 3-ml cuvettes at 25 "C by the method of Blostein and Rutter [lo], using 100 mM Tris-C1 buffer, pH 7.5, instead of glycylglycine buffer.

    All spectrophotometric measurements were per- formed using an Acta MVI recording spectrophoto- meter (Beckman).

    Analytical Procedures

    Protein concentration was determined from the absorbance at 280 nm using an A: Frn value of 9.38 [l I]. All molar concentrations were expressed per mole of aldolase unimer assuming a molecular weight of 160000 [12].

    Molecular weights were determined by Sephadex G-200 chromatography according to Andrews [13].

    Sulfhydryl group determination was carried out at 25 "C according to Ellman's procedure [14]. Total sulfhydryl content was determined in the presence of denaturing agent (2 % sodium dodecyl sulfate or 8 M urea).

    For disulfide bond determination, aldolase samples were reduced using sodium borohydride in 8 M urea under nitrogen atmosphere. After reduction the num- ber of free - SH groups was determined and compared with the amount of -SH groups determined without previous reduction [ 151.

    Determination of sulfenyl groups was carried out according to Hartman [16].

    Reduction of aldolase polymers into the unimer was performed by 48-h dialysis of the protein sample against 10 mM dithiothreitol in 100 mM Tris, pH 7.0, 1 mM EDTA.

    Spectrophotometric Monitoring of the Reaction of Diazenes with Thiols

    2.9 ml of 0.2 mM diazene derivative in 100 mM acetate buffer, pH 3.8, 1 mM EDTA was mixed with 0.1 ml 0.18 mM aldolase solution. Absorbance changes were recorded at 292 nm (absorption maxi- mum for N = N double bond) as a function of time at 20 "C against a reference sample containing diazene in buffer solution.

  • M. T. Mas, J. Buczylko, and M. Kochman 395

    I I

    Time (days)

    Fig. 3 . Reaction of diazene I with aldolase at p H 6.5. The reaction of 2.13 mM diazene I with 25.87 pM aldolase in the presence 0 1 100 mM phosphate, 1 mM EDTA, pH 6.5 at 5 "C was analyzed as a function of N = N double bond decay as A292 (M), activity loss (A--A) and aggregate formation. Open symbols represent control experiments : (&---O) A292 decrease in absence of aldolase, (A-A) activity loss in absence of diazene. The curves at the top represent Sephadex-(3-200 protein elution profile after 1, 2 and 8 days of aldolase incubation with diazene I

    1 2 3 4 5 6 7 8

    Aldoluse Modification with 1,2-Diazenedicarboxylic bis (N-methylpiperazide) , Diuzene I

    Aldolase samples in 100 mM phosphate buffer, pH 6.5, 1 mM EDTA were mixed with diazene I at 5 "C. Final aldolase and diazene concentrations were 25.9 pM and 2.13 mM, respectively. At appropriate time intervals aliquots were withdrawn from the reaction mixture and applied to a Sephadex G-25 column (2.5 x 40 cm), equilibrated with 100 mM phos- phate buffer, pH 6.5, 1 mM EDTA (sample A) or with 50 mM Mops, pH 6.5, 1 mM EDTA (sample B). Then the aliquots were assayed for enzymatic activity, sulfhydryl group content and molecular weight.

    Isolation of Aldoluse Polymers

    6mg of diazene was solubilized in 10 ml of 100 mM phosphate buffer, pH 6.5, 1 mM EDTA, 4.14 mg/ml aldolase and allowed to react at 5 "C for three days. Then the reaction mixture was applied to a Sephadex G-25 column (2.5 x 37 cm) equilibrated with 50 mM Mops, pH 6.5, 1 mM EDTA. Protein- containing fractions were collected and stored at 5C for eight days for further polymerization. Then 1.5 ml of the above solution containing 2 mg aldolase was applied to a Sephadex G-200 column (1.6 x 90 cm). Protein fractions of molecular weight 480 000 and

    higher were collected and concentrated by Amicon membrane PM 30 ultrafiltration.


    Reaction of Diuzene I with Aldoluse

    Preliminary experiments performed at equimolar concentrations of diazene and aldolase sulfhydryl groups showed the decrease of reactivity of diazenes in the order I1 > I > 111. In 100 mM phosphate buffer at pH 6.5 the reaction with diazene I1 was so rapid that within a few hours the aggregation was followed by precipitation and loss of activity. There- fore diazene I as the second most reactive agent was chosen for diazene-induced aldolase cross-linking studies.

    Fig.1 illustrates the reaction course of diazene I with aldolase in phosphate buffer at pH 6.5.measured as a function of N = N double bond absorbance at 292 nm. The ratio of diazene: thiol was equal to 5.1, assuming 16 accessible -SH groups per aldolase unimer. A 2 9 2 absorbance decrease due to the aldolase- diazene interaction can be observed within the first day of incubation. Further decrease of A292 is es- sentially due to hydrolysis of the diazene. However, a gradual decrease of activity and increase of high- molecular-weight material, mainly the dimeric frac- tion, is observed within the period of eight days.

    To investigate this process, the excess of diazene was removed from the reaction mixture by gel filtra- tion after three days of modification; this removal stops the inactivation of aldolase and also stops aggregate formation (Fig. 2, curve A). However, removal of the diazene and phosphate causes further inactivation of aldolase and aggregation leading to a material which emerges near the excluded volume upon Sephadex G-200 chromatography (Fig. 2, curve B). Resolution of the aggregate by means of Sepharose 6B column chromatography indicates polymerization of aldolase to dimer and then trimer and a small fraction of higher-molecular-weight species. The above obser- vations suggest the formation of rather stable diazene- aldolase intermediates which can react with free - SH groups exposed after removal of phosphate.

    The presence of 1 mM fructose 1,6-bisphosphate instead of 100 mM phosphate also markedly protects aldolase against further aggregation.

    In the next experiment the reaction of Nbsz with the postulated diazene-aldolase intermediate was fol- lowed. Thus the reaction of diazene I with aldolase in the presence of phosphate was allowed to proceed for three days. Afterwards, the diazene or the diazene and phosphate were removed from the reaction mixture by Sephadex G-25 column chromatography as described in Materials and Methods. Aldolase

  • 396 Use of Diazene for Cross-Linking of Aldolase

    j trimer I

    60 80 100 Elution volume (ml)


    w - - 100

    2 4 6 8 10 Time (daysj

    Fig. 2. Aldolase inactivation and aggregate formation in the reaction mixture after removal of diazene I. Aldolase and diazene I were incubated for three days at 5C in phosphate buffer, pH 6.5, as described in the legend to Fig. 1. In experiment A, a Sephadex G-25 column equilibrated with 100 mM phosphate, 1 mM EDTA, pH 6.5 was used for removal of the diazene. Approximately 6 ml of sample was applied to a 2.5 x 37-cm column. In the experiment B, the analogous Sephadex G-25 column equilibrated with 50 mM Mops, pH 6.5, 1 mM EDTA was used for removal of diazene and phosphate. Activity is expressed as a percentage of initial activity before the addition of diazene to aldolase. Time is expressed in days after diazene removal from the reaction mixture. In the control, no diazene was added to aldolase. Curve A represents the Sephadex G-200 protein elution profile 8 days after diazene removal. Curve B represents the Sephadex G-200 protein elution profile 8 days after diazene and phosphate removal from the reaction mixture

    samples containing phosphate and without phosphate were titrated with Nbsz (Fig.3, A and B respectively) at different time intervals. Titration performed im- mediately after diazene removal (sample A) revealed a decrease of 412-nm absorbance in comparison with the reference sample, indicating a decrease in the amount of thionitrobenzoate anion (Nbs). Titration performed on sample A on subsequent days showed that the amount of thionitrobenzoate released in- creased gradually up to 8 mol/mol aldolase unimer and approached a constant level for aldolase samples incubated with phosphate for 9-10 days after di- azene removal (Fig. 3A). Titrations of phosphate- free aldolase sample B performed immediately after diazene removal also showed a tendency for a decrease of 412-nm absorbance which was, however, not so pronounced as in the case of sample A. It was nine days before the amount of titratable - SH groups in aldolase reached a maximum after diazene removal. The above experiments indicate a slow decomposition of the diazene-aldolase intermediate and partial - SH group recovery.

    '* t

    Time (days)

    Fig.3. Nbsz titration of aldolase samples after removal of ( A j diazene and ( B j diazene and phosphate. Diazene I and aldolase were incubated in the presence of phosphate for three days under conditions described in Fig. 1. Then diazene (e-.), or diazene and phosphate ( 0 0 ) were removed by gel filtration, and aldolase was titrated with 5,5'-dithio-bis(2-nitrobenzoic acid) (N bsz). Squares represent the control experiment performed on aldolase sample which had not been treated with diazene. The absorbance changes were followed spectrophotometrically at 412 nm. Cal- culation of the amount of Nbs anion released was based on the measurement after 10 min of the reaction with Nbsz

    The presence of 1 - 2 sulfenyl groups/aldolase unimer can be detected just after diazene and phos- phate removal.

    Characterization of Aldolase High- Molecular- Weight Species

    The protein fraction of molecular weight 480000 and higher was isolated and analyzed for - SH group content as shown in Table 1. This fraction has an average of 13 -SH groups modified per unimer, among which six belong to 'buried' groups. Of the 13 -SH groups modified, 10 groups were restored after dithiothreitol reduction and all by borohydride reduction. Thus five disulfide bridges can be calculated on the basis of the number of -SH groups released after dithiothreitol reduction, among which two seem to be intramolecular and three intermolecular.

    Disulfide bond reduction of polymers using dithio- threitol has led to one major fraction of molecular weight 160000 and activity 15.8 units/mg protein, comparable to the activity of native aldolase unimer. Comparison of the kinetic properties of aldolase polymers and native aldolase indicates that cross- linkage of aldolase slightly affects the catalytic pro- perties. Values for fructose 1,6-bisphosphate of V = 6.4 ymol min-' mg-' and K, = 6.25 yM were found for the aldolase polymer compared to V = 15.8

  • M. T. Mas, J. Buczylko, and M. Kochman 391

    pmol min- mg- and K, = 3.4 pM for the native aldolase.

    Carboxypeptidase A digestion of aldolase polymers indicates unchanged accessibility of C-terminal func- tional tyrosine residues in comparison to that in native aldolase. The detailed analysis of kinetic and molec- ular properties of aldolase polymers will be published elsewhere.

    Application of Diazene I f o r Protein Binding to a Solid Matrix

    Two sets of experiments were performed. In the first, native aldolase was applied on diatene-activated

    Table 1. - SH-group content of aldolase polymers Aldolase polymers formed after diazene I and phosphate removal were isolated as described in Materials and Methods. They were reduced with NaBH4 in the presence of 8 M urea. The total number of -SH groups was determined in 2 % sodium dodecyl sulfate solution according to Ellmans procedure [14]. The accessible groups were determined after 15 min of reaction with Nbsz. The number of buried groups was calculated from the difference of total and accessible groups. The number of modified groups was calculated from the difference between the number of -SH groups present in native aldolase (32) and the total number of -SH groups in polymers; only 9 -SH groups were modified in aldolase polymers in the presence of phosphates

    -SH group SH groups/aldolase unimer ( M , = 160000)

    polymers reduced native aldolase polymers (control)


    Total 19 32 32 Accessible 9 15 16 Buried 10 17 16 Modified 13 0 0

    SH-Sepharose and in the second diazene-activated aldolase was applied directly on the SH-Sepharose column.

    As shown in Table 2, in both cases more than 1.5 mg aldolase can be covalently bound per ml of sedimented gel volume. The enzyme can be quanti- tatively released by reduction with dithiothreitol without loss of enzyme activity. In a control experiment no aldolase was absorbed on the free thiol form of thiopropyl-Sepharose 6B.


    In order to study protein-protein interaction via disulfide bridge formation, there is a need for mild specific reagents which oxidize sulfhydryl groups without altering protein conformation.Diazenes seem to be good reagents for this purpose.

    As shown in this paper, the reaction of aldolase with diazenedicarboxylic bisamides proceeds under mild conditions and is significantly slower than that for low-molecular-weight compounds. By analogy to low-molecular-weight compounds, this reaction may proceed in several steps [Eqns(l - 3)] [2]. The following observations seem to support the above finding. In the presence of phosphate, which protects Cys-336 and Cys-72 [17], the exposed sulfhydryl groups of residues 237 and 287 may be partially involved in aggregation, mainly dimerization, and partially in an intermediate adduct formation according to the first step of the reaction [Eqn [l)].After removal of the excess diazene I (Fig. 2, curve A) and under conditions in which Cys-336 and Cys-72 -SH groups are protected, the reaction course markedly slows down. At this stage the number of modified -SH groups as well as the enzymatic activity remain constant. Removal of phosphate

    Table 2. Aldoluse immobilization with thiopropyl-Sepharose 6 B Thiopropyl-Sepharose 6B in its free-thiol form (SH-Sepharose) was prepared by treatment with 20 mM dithiothreitol. Then free sulfhydryl groups of either Sepharose or aldolase were converted into diazene intermediates during a 1-h incubation in the presence of 10 mM or I mM, respectively, of diazene I. Both activation and coupling were performed in 100 mM phosphate buffer, pH 6.5, 1 mM EDTA, 300 mM NaCl at 4C. In a typical coupling experiment approximately 5 mg of protein was incubated with 1 ml gel volume. Unbound aldolase was then washed out and total activity and protein content determined. Immobilized aldolase was released from the solid matrix by a I-h re- duction with 20 mM dithiothreitol in 100 mM phosphate buffer, pH 8.0, 1 mM EDTA, 300 mM NaC1. Reducing agent was separated on Sephadex G-25 column before protein and activity were determined

    Experimental conditions Applied Eluted with buffer Eluted after dithio- Total amount eluted threitol reduction

    ~ -~ ~ ~ ~~~ ~ ~ ~~~ ~ ~ ~

    protein activity protein activity protein activity protein activitj

    mg U mg U mg U mg U

    Native aldolase + diazene-activated SH-Sepharose 6B s.5 91.3 4.1 61.5 1.7 28.3 5.8 89.8

    Diazene-aldolase + SH-Sepharose 6B 5.6 73.2 4.0 61.1 1.6 26.9 5.6 88.0 5.5 91.3

    Native aldolase + SH-Sepharose 6B (control) 5.2 90.7 5.2 83.5 0 0 5.2 83.5

    a Values for native aldolase.

  • 398 M. T. Mas, J. Buczylko, and M. Kochman: Use of Diazene for Cross-Linking of Aldolase

    from unimer and dimer mixtures causes uncovering of two 'protected' cysteinyl residues per subunit. Reaction of these -SH groups with those which have previously formed an intermediate adduct may occur. It is reflected in: (a) the rapid decrease of activity observed immediately after phosphate re- moval, (b) the increase of the number of modified -SH groups from 9 to 13, and (c) progress of ag- gregation leading to the formation of trimeric and higher molecular weight species.

    Disappearance of thionitrobenzoate anion during the early stages of aldolase incubation (after diazene removal) confirms the existence of a diazene-aldolase intermediate. The following reaction [Eqns (7, S)] can be involved in the decrease of 412-nm absorbance:

    mediate may be of great importance for cross-linking the protein molecules to a solid support and making immobilized enzymes.

    We thank Dr P. A. Hargrave for his helpful suggestions in the revision of the manuscript. This work was supported by Polish Ministry of Science, Higher Education and Technology, Grant R.1.9. and M.R.II.15.

    REFERENCES 1. Rasched, J. R., Bohn, A. & Sund, H. (1977) Eur. J . Biochem. 74,

    2. Kosower, E . M. & Kanety-Londner, H. (1976) J . Am. Chem.

    3. Kosower, N. S., Kosower, E. M. & Koppel, R. L. (1977) Eur,

    365 - 377.

    SOC. 98, 3001 - 300.5.

    J . Biochem. 77. 529 - 534.

    A-SNNHCOR + Nbs2 -+ A-S-Nbs I + RCONNCOR + Nbs

    COR I 1 HH

    or A-S- + Nbs2 3 A-S-Nbs + Nbs

    Nbs + A-S-NNHCOR -+ A-S-Nbs I + RCONNCOR

    COR I I HH

    4. Srivastava, S . K., Awasthi, Y. C. & Beutler, E. (1974) Biochem.

    5. Harris, J. W. & Biaglow, J. E. (1972) Biochem. Biophys. Res.

    6. Kosower, E. M., Correa, W., Kinon, B. J. & Kosower, N. S. (1972) Biochim. Biophys. Acta, 264, 39-44.

    7. Haest, C. W. M., Kamp, D., Plasa, G. & Deuticke, B. (1977) Biochim. Biophys. Acta, 469, 226- 230.

    8. Srivastava, S. K., Awasthi, Y. C. & Beutler, E. (1974) Biochem.

    9. Penhoet, E. E., Kochman, M. & Rutter, W. J. (1969) Bio-

    10. Blostein, R. & Rutter, W. J. (1963) J . Bid. Chem. 238, 3280-

    J . 139,259-215.

    Commun. 46, 1743- 1749. (7)

    J . I39,289-295.

    (8) chemistry, 8, 4391 -4395.

    3285. 1 1. Donowan, W. (1964) Biochemistry, 3, 67 - 73. 12. Kawahara, K. & Tanford, C. (1966) Biochemistry, 5, 1578- where A = aldolase and R = N(CNz)dNCH3.

    The above results indicate that diazene derivatives 1584 1 _ - ..

    may be used as a to01 for probing the reactivity and availability of Some protein thiol groups. These 14. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. compoun~s might also be of importance in the study

    13. Andrews, P. (1965) Biochem. J , 96, 595-606.

    1.5. Cavallini, D., Graziani, M. T. & Dupre, S. (1966) Nature (Lond.) 212,294-295.

    Of proteins in which sulfhydryl groups and disulfide 16. Hartman, F. C, (1970) Biochemistry, 9, 1783-1791. bridges are known to play an important physiological 17. Lai, C. Y., Nakai, N. & Chang, D. (1974) Science (Wash. role. The existence of a stable diazene-protein inter- 0.C.j I83, 1204- 1206.

    M. T. Mas, J. Buczylko, and M. Kochman*, Zespo1 Biochemii, Instytut Chemii Organicznej i Fizycznej, Politechnika Wroclawska, Wybrzeie Wyspianskiego 27, PL-50-370 Wrochw, Poland

    * To whom correspondence should be addressed.


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