the use of diazenedicarboxylic acid derivatives for protein cross-linking : use of diazene for s-s...
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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 . 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 184.108.40.206). 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
RS-NNHCOR+ RS --+ R-S-S-R+ RCONNCOR (2) I I I
RCONNHCOR+ H20-+RrCO%NHCOR f R-SdH2 I S (3) I R
R-SdH2 -+ R-SOH + H + (4)
( 5 )
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.  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 , very little effect on the activity of some glycolytic enzymes  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.
MATERIALS AND METHODS
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 . All reagents were of analytical reagent grade. All solutions were prepared in deionized water.
Rabbit muscle aldolase was prepared according to Penhoet et al. . 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).
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 .
Molecular weights were determined by Sephadex G-200 chromatography according to Andrews .
Sulfhydryl group determination was carried out at 25 "C according to Ellman's procedure . 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 .
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
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,