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

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<ul><li><p>Eur. J. Biochem. 100, 393-398 (1979) </p><p>The Use of Diazenedicarboxylic Acid Derivatives for Protein Cross-Linking Use of Diazene for S-S Cross-Linking of Aldolase </p><p>Maria T. MAS, Janina BUCZYLKO, and Marian KOCHMAN </p><p>Department of Biochemistry, Technical University of Wroclaw </p><p>(Received May 16, 1979) </p><p>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 &gt; I &gt; 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. </p><p>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. </p><p>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. </p><p>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: </p><p>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. </p><p>Enzyme. Aldolase (EC Note. The term unimer has recently been proposed for use in </p><p>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. </p><p>R-SH + R-C-N = N-C-R -+ R-C-N-N-C-R (1) I I </p><p>I H+ R </p><p>S H </p><p>RS-NNHCOR+ RS --+ R-S-S-R+ RCONNCOR (2) I I I </p><p>COR HH </p><p>RCONNHCOR+ H20-+RrCO%NHCOR f R-SdH2 I S (3) I R </p><p>R-SdH2 -+ R-SOH + H + (4) </p><p>( 5 ) </p><p>(6) </p><p>0 I/ </p><p>R-SOH + R-SOH -+ R-S-S-R + H20 R-S-S-R + H20 -+ R-SH + R-S-OH </p><p>II II 0 0 </p></li><li><p>394 Use of Diazene for Cross-Linking of Aldolase </p><p>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 </p><p>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]. </p><p>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. </p><p>[Eqn (4 - 6)1 PI. </p><p>MATERIALS AND METHODS </p><p>Reagents and Enzymes </p><p>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. </p><p>Diazenedicarboxylic acid bisamides : 1,2-diazene- dicarboxylic bis(N-methylpiperazide) (I) 1,2-diazene- dicarboxylic acid bis(N,N'-dimethylpiperazide) diio- </p><p>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. </p><p>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. </p><p>All spectrophotometric measurements were per- formed using an Acta MVI recording spectrophoto- meter (Beckman). </p><p>Analytical Procedures </p><p>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]. </p><p>Molecular weights were determined by Sephadex G-200 chromatography according to Andrews [13]. </p><p>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). </p><p>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. </p><p>Determination of sulfenyl groups was carried out according to Hartman [16]. </p><p>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. </p><p>Spectrophotometric Monitoring of the Reaction of Diazenes with Thiols </p><p>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. </p></li><li><p>M. T. Mas, J. Buczylko, and M. Kochman 395 </p><p>I I </p><p>Time (days) </p><p>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 : (&amp;---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 </p><p>1 2 3 4 5 6 7 8 </p><p>Aldoluse Modification with 1,2-Diazenedicarboxylic bis (N-methylpiperazide) , Diuzene I </p><p>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. </p><p>Isolation of Aldoluse Polymers </p><p>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 </p><p>higher were collected and concentrated by Amicon membrane PM 30 ultrafiltration. </p><p>RESULTS </p><p>Reaction of Diuzene I with Aldoluse </p><p>Preliminary experiments performed at equimolar concentrations of diazene and aldolase sulfhydryl groups showed the decrease of reactivity of diazenes in the order I1 &gt; I &gt; 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. </p><p>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. </p><p>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. </p><p>The presence of 1 mM fructose 1,6-bisphosphate instead of 100 mM phosphate also markedly protects aldolase against further aggregation. </p><p>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 </p></li><li><p>396 Use of Diazene for Cross-Linking of Aldolase </p><p>j trimer I </p><p>60 80 100 Elution volume (ml) </p><p>Control </p><p>w - - 100 </p><p>2 4 6 8 10 Time (daysj </p><p>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 </p><p>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. </p><p>'* t </p><p>Time (days) </p><p>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...</p></li></ul>