Reactivity of aromatic and heterocyclic disulphides with thiol group of bovine serum albumin

Reactivity of aromatic and heterocyclic disulphides with thiol group of bovine serum albumin Martine Gossdet*, Jean-Pierre Mahieu and Bernard Sebille Laboratoire de Physico-Chimie des Biopolym~res, Universit~ Paris Val de Marne, Cr~teil 94000, France (Received 10 September 1987; revised 21 December 1987) The reactivities of several aromatic and heterocyclic disulphides with thiol groups of bovine serum albumin were determined at physiological pH 7.0 or 7.4 by two methods: spectrophotometry and chromatography. The apparent second-order rate constants k I were calculated. It was shown that the two five-membered heterocyclic disulphides examined, the 2,2'-dithiodiimidazole and the 3,3'-dithiodi(1 H-1,2,4 triazole) are more reactive than the six-membered compounds. The lipophilie properties of the nine disulphides were evaluated by reversed-phase high performance liquid chromatography. The method consisted in the determination of the percentage of acetonitrile (Pac) required for eluting each compound in water-acetonitrile gradient. The structure-reactivity correlation between log k x and log P Ac was established, and fitted to a parabolic curve, leading to the conclusion that maximum reactivity corresponds to a narrow domain of disulphide lipophilicity. Keywords: Bovine serum albumin; disulphides; lipophilicity; structure-reactivity relationships

Introduction The thiol-disulphide interchange reaction has largely been studied ori small molecules and on proteins. The most commonly used disulphide for quantitative analysis of protein thiol groups was developed by Ellman t'2: 5,5'dithiobis(2-nitrobenzoic acid) symbolized by ESSE hereafter. However the range of pH at which this reagent can be effectively used is limited since below pH 7.0 no reaction occurs. Determinations o f - S H values were usually carried out between pH 7.8 and 10.4 (Refs 3-5). Similar reagents especially 2,2'-dithiodipyridine (2PDS) and 4,4'-dithiodipyridine (4-PDS) were used for --SH groups analysis in proteins4'6-s. Another disulphide, the 6,6'-dithiodinicotinic acid 5 was less commonly used with proteins, due to its weak reactivity in neutral medium. All these compounds react with bovine serum albumin (BSA) free -SH groups to give the mixed disulphide and the corresponding thione or thiol. It is therefore possible to follow the reaction course by spectroscopy in the case where the thione has an absorption distinct from that of the disulphide. In the present work we have measured the reactivities of these usual disulphides and of some new heterocyclic ones on the -SH groups of BSA. Further, in order to set up a structure-reactivity correlation, we have determined the hydrophobic character of each disulphide from a chromatographic analysis.

benzoic acid) was from Jansen, but we prepared the dipotassium salt according to Wilson et al. 3. Other disulphides were purchased from Aldrich (2mercaptopyrimidine), from Jansen (2-mercapto-4methyl-pyrimidine hydrochloride and 1 H-l~2,4-triazole3-thiol), from Ventron (2-mercaptoimidazole). 2Mercaptopyridine-N-oxide sodium salt and its disulphide were from Goldschmidt France. Iodine, sodium hydrogeno carbonate, ammonium persulphate were from Prolabo.

2,2'-Dithiodipyrimidine, 2,2'-dithiodi(4-methylpyrimidine) and 3,Y-dithiodi (1 H-I~2,4 triazole). Were

Ex~rimenml Materials

obtained by oxidizing the corresponding thiol with iodine in alkaline aqueous solution or in alkaline water-ethyl alcohol mixture according to a classic procedure9-1~. The reaction was conducted at room temperature except for the 2-mercapto-4-methylpyrimidine water-ethyl alcohol solution which was oxidized at 50°C. The disulphides thus obtained were washed and recrystallized. The yield for 2,2'-dithiodipyrimidine was 77 9/0,melting point 142°C. Anal. calc. for CsH6N4S2 : C, 43.22; H, 2.69; N, 25.2; S, 28.65. Found: C, 42.99; H, 2.62; N, 25.26; S, 29.03. The yield for 2,2'-dithiodi(4-methylpyrimidine) was 40 %, melting point 109°C. Anal. calc. for CloHtoN4S2: C, 49.56; H, 4.16; N, 23.12; S, 26.46. Found: C, 48.35; H, 3.98; N, 22.13; S, 26.34. The yield for 3,Y-dithiodi (1 H-1 ~.,4 triazole) was 59 %, melting point 243°C. Anal. calc. for C4H4N6S2: C, 23.99; H, 2.01; N, 41.97; S, 32.02. Found: C, 24.24; H, 1.97; N, 41.28; S, 32.61.

Bovine serum albumin (lot 49 C-0441, fraction V) was purchased from Sigma. 2).'-Dithiodipyridine, 4,4'dithiodipyridine, 6,6'-dithiodinicotinic acid were from Aldrich. The Ellman's reagent 5,5'-dithiobis(2-nitro-

2,2'-Dithiodiimidazole. Was prepared by oxidation of 2-mercaptoimidazole with an ammonium persulphate aqueous solution according to a procedure previously described t 2 for another disulphide.

0141-8130/88/040241-07503.00 © 1988 Butterworth & Co. (Publishers) Ltd

Int. J. Biol. Macromol., 1988, Vol. 10, August

241

Reactivity of disulphides with BSA." M. Gosselet et al. The yield was 46 %, melting point 168°C. Anal. calc. for C6HrN4S2: C, 36.34; H, 3.0; N, 28.26; S, 32.34. Found: C, 36.07, H, 3.24; N, 27.16; S, 30.95. 2,2 '-di thiodipyr'idiDe (I)

Spectroscopic measurements Water, freshly distilled and potassium phosphate buffer (510-3M) containing EDTA (10-3M) were used throughout after nitrogen bubbling. Enough KCI was added to obtain the same ionic strength (0.02) in all the experiments. Concentrated BSA and RSSR solutions were prepared just before the experiments, and allowed to equilibrate at 25°C. Mixing of the solutions was realized with an SFA l l-Hi-Tech (Salisbury, England) rapid kinetics accessory. The reaction was monitored by measuring the appearance of thiol at its absorption maximum or, in the case of 2,2'-dithiodiimidazole, by measuring the decrease of absorbance at 300 nm. A Perkin-Elmer 550 SE UV spectrophotometer coupled to an R 100 recorder was used. Due to the low solubility of most disulphides, the concentrated solutions were prepared in phosphate buffer in the presence ofDMSO, 1 to 2 % (v/v). It was previously shown that DMSO under these conditions had no effect on the SH haemoglobin, RSSR exchange reactions ~3, or enzyme, heterocyclic disulphide exchange reactions ~4.

4,4 ' -di thiodipyridine (If)

2,2'-dithiodipyrimid/ne (~zr)

O-

CH~-S-S~CH ~ 2,2'-di~iodi(4-methy]pyrimidine) (~v)

O-

2,2'-dithiodiimidazole

(VI)

2,2'-dithiod] (pyPidine--N-o×ide) (V)

3,3'-dithiodi triazo]e)

5,5'-dithiobis

(IH-1,2,4 (VII)

(2-nitrobenzoic (V LiI)

acid)

HOgaNI.~S._SO cOzH 6,6 '-di thiodinieotinic (IX)

acid

Methods Chromatographic measurements of the kinetic rate constants Materials. Two 6000A pumps from Waters Assoc. (Milford, MA, USA) were monitored with a Waters Assoc. Model 660 solvent programmer gradient and connected to a Rheodyne (Berkeley, CA, USA) 7125 injector. The injection volume (loop) was 50/A. The column (5 cm x 7 mm ID) was filled in our laboratory with Synchropak (Synchrom, Linden, USA) AX-300 anion exchanger (pore diameter 300 A, bead diameter 10 #m). A Pye Unicam (Cambridge, UK) Type LC3 detector was used for the absorbance measurements at 280 nm. Procedure. The reactivity rate of disulphide compound with BSA was studied in 20 mu Tris buffer (pH 7.4) at 25°C in a water-bath shaker. For each reaction time a reaction vessel was filled. A sample of 50 #l of each mixture was injected into the chromatographic system. The time needed for sample transfer and injection was less than 10 s. The chromatographic column was maintained at 25°C with a thermostically regulated jacket. The flow rate was 2 ml/min.

Kinetic rate constants determination The chromatographic kinetic rate constants were determined by two methods, depending upon the presence or not of an ionic group on the disulphide of interest. The separation of the modified BSA from the unmodified one was done on an anionic exchange column. These two methods were previously described for the study of haemoglobin reactivity 15. For the determination of the kinetic constants, we have considered the following equation: kl

P-SH+RS-SR

~ P-S-SR+RSH

(1)

k- 1

A

B

C

D

RSSR is always in excess, so that, for short reaction times, the reversed reaction can be neglected. It was previously shown that this reaction was of second order so that the equation rate can be approximated to: d[RSH] dt

k , [ R S S R ] [ P - SH]

(2)

After integration and inserting initial concentrations:

Chromatographic properties of the disulphides The determination of the PAc parameters was performed with the same chromatographic equipment, i.e. two 6000 A pumps from Waters Assoc. monitored with a Waters Assoc. Model 660 solvent programmer gradient. A Nucleosil C18 (Altex, Berkeley, CA, USA) column (25 cm x 4.2 mm ID) was used, thermostatically regulated at 25°C. An Altex pump was used for the determination of the capacity factors. In this case the column was a Lichrosorb RP18 Merck (10#m) (25cm x4.6mm ID); the flow-rate was fixed at 1.53 ml/min.

242

Int. J. Biol. Macromol., 1988, Vol. 10, August

[At] = [Ao] - [X] [8,] = [80] - [x] [Ct] = [/)t] = IX]

1 lo- [B°] - [X]k t a_rte [Bo] -~ [Ao] g~ [Ao] - IX] ' " ' ~ 1 [Ao] [ B o ] - [ X ] = k , t [Bo] - [Ao] ,og~ ~ x [Bo] _ [X] ,

Here [-A0], [Bo] are the intial concentrations [X] is known from the measured absorbance.

Reactivity of disulphides with BSA: M. Gosselet et al. Lipophilicity evaluations Several authors have proposed various approaches to quantify the lipophilicity of chemical agents. Lipophilicity was usually quantified on the basis of the logarithm of 1-0ctanol/water partition coefficients 16-19. Thin-layer chromatography (t.l.c.) 2°'21 and reversedphase high performance liquid chromatography (r.p. h.p.l.c.) are an alternative approach to the static method for the determination of partition coefficients22-26. Linear relationships between the logarithm of the capacity factor, log kl and log P~t are generally obtained for compounds of a homologous series 27-32. The lipophilic character of the disulphides was evaluated by determining the per cent of acetonitrile (P~) necessary to elute out of an RP~s reversed-phase chromatography column, the various compounds in an acetonitrile-water gradient. This method has also been used in a similar work 3a.

0

L_

,II

Results

Spectroscopic results

300

~.(n m)

350

400

Figure 1 Ultraviolet absorption spectra of thiols and disulphide. II, 2-Mercaptopyrimidine 5 10- 5 M; V, 4-Methyl-2mercaptopyrimidine 310-SM; ©, 2-Mercaptopyridine-Noxide 2 10- 5 M; , 2,2'-Dithiodiimidazole 2 10- 5 M. Experimental conditions: 0.005M phosphate buffer, 0.001 M EDTA, pH = 7.0, T = 25°C

For longer reaction times, the reversed reaction cannot be neglected and the rate is given by equation (3): d[RSH]

dt

-

diD]

dt =kt[At][Bt]-k-l[Ct][Dt]

(3)

[At], [Bt], [Ct], [Dr] represent instant concentrations I-Ae], [Be], [ C J , [De] represent concentrations at equilibrium. The complete calculations were previously published 15 and led to the expression:

log~(e+xd_c)=-k~, d. t + l o g ~ ( e - ! )

c = [Ao] - [Ae] = x - y

a ---=1

e-k 1

(4)

Table 1 Ultraviolet absorbance of thiols or disulphide in phosphate buffer (5 10- 3 M), EDTA 10- 3 M at pH 7.0

where

d=C= kl

Ultraviolet absorption spectra of thiols and disulphides were recorded (Figure I) and the absorption coefficients of these compounds were determined (Table I). For the commonly used ESSE and 6',6-dithiodinicotinic acid the respective absorption coefficients e412 = 13 600 1 mo1-1 cm -1, %60 = 10000 1 mol -~ crn -1 were used in our calculations. Most of the thiols or thiones examined presented a maximum u.v. absorbance at a wavelength higher than 300 nm, where the absorbance of BSA is negligible, except for the 2-mercaptoimidazole. However the corresponding 2,2'-dithiodiimidazole u.v. spectrum presents a shoulder at 300 nm which makes it possible to follow the reaction course by measuring the decrease of this absorbance at 300 nm. It was difficult to determine with accuracy the reaction kinetic parameters. Most disulphides underwent an hydrolytic decomposition at 25°C and neutral pH. This spontaneous decomposition induced a change in absorbance which was neglected for short reaction times, but subtracted for the others. The reactivity of the 3,3'-dithiodi(l H-1,2,4 triazole) could not be studied by this spectroscopic method because both disulphide and the corresponding thiol do not present a characteristic absorbance beyond 300 nm. The spectroscopic titration of the free sulphydryl groups of BSA was obtained according to Grassetti and Murray 4 by reacting BSA with 2-PDS or 4 PDS in large excess. The values thus measured were 0.58 and 0.60 respectively, close to the commonly used sulphydryl content (0.60 to 0.70 -SH group per BSA molecule). According to the reactivity of the disulphide with the

-

[

+k-1 ] [Ae] + [Be] kl (ICe] + [De]) k_ 1

kI

Thiol or disulphide

e

2 (nm)

2-Mercaptopyrimidine 4-Methyl-2-mercaptopyrimidine 2-Mercapto-pyridine-N-oxide 2,2'-Dithiodiimidazole

1550 2400 5560 5620

330 336 330 300

Int. J. Biol. Macromol., 1988, Vol. 10, August

243

Reactivity o f disulphides with B S A : M . Gosselet et al. Table 2

Apparent second-order rate constants for the reaction between BSA and different disulphides kl a (1 m o l - 1 s - 1)

Disulphide

I II III IV V VI VII VIII IX

160_ 10 440+ 10 28 + 2.5 4.3 ___0.4 75___7.5 2300___ 150 0.85 + 0.1 1.5 + 0.1

k_ 1a (1 m o l - t s - ~)

klb (1 m o l - 1 s- l)

k_ i b (1m o l - 1 s - 1)

200* 125-180"*

567 + 50 817+83 45 + 3 12 +0.8 113__+5 2067 + 186 2633 +237 58.4 + 3.4 21.4 + 1.7

0.1 -t-0.1

170.6 + 15 357.8 + 30

k]t

2.7+0.2 2.5+0.2

3.3***

Spectrophotometric determination at pH 7, ionic strength = 0.02, phosphate buffer 5 10 3 M, temperature 25°C, with [BSA] = 5 10- 5 M, [RSSR] = 210- 4 M, except for I, VI where [RSSR] = 10- 4 M and VIII, IX where [BSA] = 9.6 10- 6 M, [RSSR] = 5.3 10- 4 M b Chromatographic determinations at pH 7.4, ionic strength = 0.02, Tris buffer 2 10 -2 M with [BSA] = 10-5 M, [RSSR] = 2 10 -4 M, except for VI and VlI where [-RSSR]= 2 10 -~ M. Eluant, gradient from 10% to 90% B in 15 min; A, 0.02 M phosphate buffer pH 6.4; B, 0.02 Mphosphate buffer pH 6.4 with 0.3 M sodium acetate; flow rate 2 ml/min; temperature 25°C * Apparent second-order rate constant 7 at 25°C, pH 7, phosphate buffer 0.1 M ** Apparent second-order rate constant s at pH 7, phosphate buffer 0.1 M *** Apparent second-order rate constant 3 estimated at pH 7, at 25°C, ionic strength= 0.02, Tris buffer 0.005 u

- S H g r o u p s of BSA, the first or the s e c o n d m e t h o d of c a l c u l a t i o n was applied. In the first case, the k 1 rate c o n s t a n t for a given d i s u l p h i d e is o b t a i n e d b y p l o t t i n g lo

~'~4 0 vm X

=E

[A°] [B°]-[X] versus t i m e g¢ ~ x [A0] _ [ X ]

e-

This p l o t is linear for t h e first s e c o n d s with a c o r r e l a t i o n coefficient greater than 0.995 (eight to ten d e t e r m i n a t i o n s ) . In the second case a n d from a m i n i m u m o f ten e x p e r i m e n t a l m e a s u r e m e n t s , we have f o u n d t h a t the c o r r e l a t i o n coefficients of

0 om

*,3 et.1 0

u2 log~ e +

versus time were greater t h a n 0.995 X--C I,U

T h e c o n s t a n t s k~, k_ 1 were d e t e r m i n e d at p H 7.0 a n d a r e listed in Table 2. W e have tried to a p p l y the c o m p e t i t i v e m e t h o d i n t r o d u c e d b y W i l s o n a n d H u p e a to the case of nonc h r o m o g e n i c d i s u l p h i d e s a n d also for the p r e v i o u s l y studied d i s u l p h i d e s , b y using the s t a n d a r d ESSE. Figure 2 shows the c h a n g e in E S - c o n c e n t r a t i o n , as a function of time. W e have noticed, in all experiments with a r o m a t i c , heterocyclic d i s u l p h i d e s a m a x i m u m of the a b s o r b a n c e with time. T h e reactions involved are: kl

P - S H + R S S R ~- P - SSR + R S H excess

(5)

k- 1 k2

P - S H + E S S E ~ P - SSE + E S H excess

(6)

k_ 2 k3

R-SH+ESSE

~ RS-SE+ESH

(7)

k- 3 k4

E S H + R S S R ~ ES

-

SR + RSH

(8)

11)

2'0

31)

Time (rnin)

4'0

Figure2 Competitive reactions: the plot demonstrates a decrease in the E S - product ion when an aromatic or a heterocyclic disulphide is added. Experimental conditions: 0.005 M phosphate buffer, 0.001 M EDTA, pH = 7.57; [BSA] = 9.610-6M, [ E S S E ] = 1 0 . 6 1 0 - 4 M, [ R S S R ] = 1 0 - a M ; T = 25°C; ionic strength=0.02. ©, ESSE; A, ESSE and 2,2'dithiodipyridine, D M S O 0.5 %; A, ESSE and 3,3'-dithiodi(1 H1,2,4 triazole D M S O 0.5%; e , ESSE and 2,2'-dithiodi(4methylpyrimidine D M S O 0.5 %; II, ESSE and 2,2'dithiodipyrimidine D M S O 0.5%; C2, ESSE and 2,2'dithiodi(pyridine-N-oxide) DMSO 0.5 %

k- 4 ks

E S S E + R S S R ~ 2ES

-

SR

(9)

k- 5

R e a c t i o n (9) is the result of e q u a t i o n s (7) a n d (8). In W i l s o n ' s studies, r e a c t i o n (8) was neglected, d u e to the low reactivity o f RSSR. W e have f o u n d t h a t for the

244

Int. J. Biol. M a c r o m o l . , 1988, Vol. 10, A u g u s t

c o m p o u n d s studied here, this reaction does occur quickly a n d c o m p l e t e l y d u e to their g r e a t nucleophilicity. F o r e x a m p l e we have o b s e r v e d t h a t the mixing of ES - 10- 5 M with 2 2 ' - d i t h i o d i p y r i m i d i n e 1 0 - 4 M resulted in the i n s t a n t a n e o u s d i s a p p e a r a n c e of the characteristic yellow

Reactivity of disulphides with BSA: M. Gosselet et al. TaMe 3 Characterization of the hydrophobic properties of

different disulphides, obtained by varying the acetonitrile percentage P ~ in the eluant, for complete elution eAc

Disulphide

(%)

log PA¢

I II III IV V VI VII VIII IX

77 81 71 78 40 59 57 34 37

1.886 1.908 1.851 1.892 1.602 1.771 1.756 1.531 1.568

The dution is done on a Nuclvosil Cls (25 c m x 0.42 cm ID) column. The gradient used was H~O 100% to acvtonitrile 100% for 15rain I : 2-PDS II : 4-PDS III : 2~.'-dithiodipyrimidine IV : 2,2'-dithiodi(4-mvthylpyrimidine) V : 2,2'-dithiodi(pyridine-N-oxide) VI : 2,2'-dithiodiimidazolc VII : 3,3'-dithiodi(1 H-1,2,4 triazole) VIII : ESSE IX : 6,6'-dithiodinicotinic acid

colour of E S - . This is why this method was not used for determining the kinetic constants.

Chromatographic results The choice between the two methods described above for the determination of the kinetic constants depended upon the charge of RSSR. These two methods were especially useful in the case of high reaction rates. The results are listed in Table 2. Measured values of P ~ required to elute out of an RPIa reversed-phase chromatography column for various compounds are reported in Table 3. Values of log k 1 plotted versus log PA~ are shown in Figure 3. A maximum of log kl appears in a narrow domain of log PAc values. The overall shape of the curve is parabolic. We have determined the coefficients a, b, c of the second-order equation (10) after minimization with a computer: log PA¢= a(log p)2 + b(log P) + c

(10)

and found a= -53.39, b= 187.594, c= -162.025 for the spectroscopic experiments at pH 7, with a correlation coefficient am=0.77, and a=34.517, b=-120.48, c= - 102.197 for the chromatographic experiments at pH 7.4 with a correlation coefficient a m= 0.682.

4

a

b pH "/.4

pH 7.0 aVll 3 -II .t¢

.¢

2

¢1) O ,.I

0 _1

III

2 []

III

olX Q IV

IV VIII =IXI 1.6

I

I

t8

I

I

i

2

1.6

1.8

2

Log PAc

Log

PAc

Fignre 3 Structuro-reactivity correlation between the apparent rate constant for the reaction of disulphides with the thiol group of BSA and the logarithm of the acvtonitrile percentage. The upper plots (a) were obtained from apparent rate constants determined by spectroscopy at pH 7.0in 0.005 M phosphate buffer; 0.001 M EDTA; ionic strength = 0.02; T = 25°C. The lower plots (b) were obtained from apparent rate constants determined by a chromatographic method at pH 7.4 in 0.02 M Tris buffer; ionic strength = 0.02; T = 25°C

Int. J. Biol. Macromol., 1988, Vol. 10, August

245

Reactivity of disulphides with BSA: M. Gosselet et al. Table 4 Experimental and literature pKa values of thiols. The experimental values were determined under nitrogen for dilute solutions of thiols (10-3 M) at 20°C Thiol 2-Mercaptoimidazole 2-Mercaptopyridine 4-Mercaptopyridine 4-Methyl-2-mercaptopyrimidine 2-Mercaptopyrimidine 1 H-1~2,4 triazole thiol 6-Mercaptonicotinic acid 2-Mercaptopyridine-N-oxide 5-Mercapto-2-nitrobenzoic acid

pK a (exp.)

7.96 7.35 7.37

pK a (Refs) 11.6 (34) 9.81 (35,36) 8.65 (35,36) 8.1 (37) 7.10 (37) 6.54 (38)

4.95 4.5 (39)

,,VII

-3 ,=~ ell o •-i 2

,,VI ,,11

.V *VIII

,,I

,,111 ,,IX ,IV

Discussion

The k I values reported in Table 2 show a general agreement between the two experimental methods although the kl values obtained for compounds VIII and IX by chromatography are much higher than those obtained by spectroscopy. It must be noted that the spectroscopic results are in good agreement with those of Wilson at the same pH (Ref. 3). We have verified that the stirring of the reaction mixture, which was used before chromatographic measurements, had no incidence on the k 1 values. For compounds VIII and IX, the reciprocal k_ 1, determined by spectroscopy at pH 7 are high, 170.6 1 mo1-1 s -1 and 357.81 mo1-1 s -1 respectively. For compounds I to VII, the slight difference of pH conditions (ApH=0.4) explains the variation of the kinetic constant. From the results of Pedersen 8, a factor 2 is predictable in the case of the reaction of 2-PDS with BSA. For this reagent the spectroscopic value agrees with that of Svenson 7 and Pedersen s. The highest apparent rate constants were found for the two heterocyclic disulphides, the 2',2'-dithiodiimidazole and the 3,3'dithiodi(1 H- 12,4 triazole). The pK a values for the thiols are listed in Table 4. Figure 4 shows that no correlation exists between the pKa of the thiols and the rate constants. However for a set of compounds, an increase of the pK a decreases the rate constant as predicted 39-42. This applies to 2 and 4-PDS and to III and IV pyrimidine derivatives. Indeed the 4-mercaptopyridine is a stronger acid than the 2-mercaptopyridine, and hence a better leaving group like the 2-mercaptopyrimidine compared with the 4-methyl2-mercaptopyrimidine. In the series of pyridine compounds, when the pyridine thiol compound contains a polar portion this does not hold; that is the case for 2,2'dithiodi(pyridine-N-oxide) and 6,6'-dithiodinicotinic acid. This fact suggests that the lipophilic character plays a role in the reactivity as will be discussed below. Figure 3 shows that there is an optimum log Pac value, beyond which the reactivity, or log kl, decreases. This optimum PAt corresponds to the most reactive compounds VI and VII, the 2,2'-dithiodiimidazole and the 3,Y-dithiodi(1H-1,2,4 triazole). This correlation shows that the six-membered heterocyclic disulphides of the pyridine group are all the more reactive as their hydrophobic character increases: the 2- and 4-PDS are more reactive than the 2,2'-dithiodi(pyridine-N-oxide) or the very hydrophilic 6,6'-dithiodinicotinic acid. These observations are consistent with studies which conclude

246

Int. J. Biol. Macromol., 1988, Vol. 10, August

I

(0

s P Ka Figure 4

The plots show that there is no correlation between

the apparent rate constant and the pK a of the thiol released for the reaction of disulphides with the thiol group of BSA that the thiol group of the BSA is in a sterically restricted environment that has hydrophobic character 3. This structure-reactivity correlation is similar to those reported by Hansch 4a for the binding of drugs to proteins or for the biological activity of drugs. Indeed Hansch stated a relationship between log l/C, where C is the molar concentration of a drug producing standard response in constant time, and log P, where P is the octanol-water partition coefficient. This relationship fitted best with a parabolic expression such as equation (11): pC(k) = - a(log p)2 + b log P + constant

(11)

a, b, are constants; other rate equilibrium constants (k) may also be used. In our work, the PAc parameter, easy to measure reflects the solubility properties of a given compound in the organic layer of the stationary phase. The experimental values of log PAc correlated to the logarithm of the kinetic k 1 constants by a quadratic relationship let us consider the PA~ parameter as a lipophilic parameter, comparable to the octanol-water partition coefficient used by Hansch. The knowledge of this PA~ parameter permits us to stress the best structural conditions for a high biological activity. The great reactivity of the five-membered heterocyclic ring with enzymes has already been pointed out by Schmidt et al. 14 and was attributed to the small size of these compounds. Our results confirm these conclusions but let it appear further that their great reactivity corresponds to a medium hydrophobic character. References

1 2 3

Ellman,G. L. Arch. Biochem. Biophys. 1959,82, 70 Pfleiderer,G., Holbrook, J. J., Nowicki, L. and Jeckel, D. Biochem. Z. 1966, 346, 297 Wilson,J. M., Wu, D., Motiu de Grood, R. and Hupe, D. J. J. Am. Chem. Soc. 1980, 102, 359

R e a c t i v i t y o f disulphides with B S A : M . Gosselet et al.

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Grassvtti, D. R. and Murray, J. F. Arch. Biochem. Biophys. 1967, 119, 41 Podhradsky, D., Kristian, P. and Novotny, L. Biology 1982, 37(12), 1141 Kail, M., Schnvid©r,F. and Wvnek, H. Z. Physiol. Chem. 1970, 351, 1280 Svvnson, A. and Cadsson, J. Biochim. Biophys. Acta, 1975,400, 433 Pederscn, A. O. and Jacobsen, J. Fur. J. Biochem. 1980,106,291 Ghosh, T. N. and Guha, P. C. J. Ind. Chem. Soc. 1929, 6, 193 Grassetti, D. R., Murray, J. F., Brokke, M. E. and Gutman, A. D. J. Med. Chem. 1967, 10(6), 1170 Goerdeler, J. and Galinke, L Chem. Bet. 1957, 90, 202 D'Amico, J. J. J. Am. Chem. $oc. 1953, 75, 102 Gar¢l, M. C., Beuzard, Y., ThiUvt,J., Domenget, C., Martin, J., Galacteros, F. and Rosa, J. Eur. Biochem. 1982, 123, 513 Schmidt,U., Pfleidervr,G. and Bartkowiak, F. Analyt. Biochem. 1984, 135, 217 Mahieu, J. P., Sebille, B., Gosselet, M., Garel, M. C. and Bvuzard, Y. J. Chromatogr. 1986, 359, 461 Overton, E. Z. Phys. Chem. 1897, 22, 189 Meyer, H. Arch. Exp. Pathol. Pharmakol. 1899, 42, 109 Hansch, C., Unger, S. H. and Forsythe, A. B. J. Med. Chem. 1973, 16, 1207 Hansch, C. in 'Drug Design', (Ed. E. J. Aliens), AcademicPress, New York, 1971, Vol. 1, p. 271 Boyce,C. B. C. and Milborrow, B. V. Nature 1965, 208, 537 Tomlinson, E. J. Chromatogr. 1975, 113, 1 Haggerty,W. J. and Murril, E. A. ResiDer. 1974, 25, 30 McCall, J. M. J. Med. Chem. 1975, 18, 549 Carlson, R. M., Carlson, R. E. and Kopperman, H. L. J. Chromatogr. 1975, 107, 219

25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43

Chen, B. K. and Horvath, C. J. J. Chromatogr. 1979, 171, 15 Nahum, A. and Horvath, C. J. Chromatogr. 1980, 192, 315 El Tayar, N., Van de Waterbo3md, H. and Testa, B. J. Chromatogr. 1985, 320, 320 Miyake, K., Mizuno, N. and Terada, H. Chem. Pharm. Bull. 1986, M(11), 4787 Valko, K. J. Liquid Chromatogr. 1984, 7(7), 1405 Yamagami, C., Takami, H., Yamamoto, K., Miyoshi, K. and Takao, N. Chem. Pharm. Bull. 1984, 32, 4994 Yamana, T., Tsuji, A., Miyamoto, E. and Kubo, O. J. Pharm. Sci. 1977, 66, 747 Butte, W., Fooken, C., Klussmann, R. and Schuller, D. J. Chromatogr. 1981, 214, 59 Garel, M. C., Domenget, C., Bcuzard, Y. and Mahieu, J. P., Gossdet, M. and Sebille, B. Eur. J. Biochem. (submitted) Stanovik, B. and Tislor, M. Anal. Biochem. 1964, 9, 68 Albert, A. in 'Physical Methods in Heterocylic Chemistry', (Ed. A. R. Katritsky), AcademicPress, New York, 1963, eel. 1, p. 1 Brokleshurst,K. and Little, G. Biochem. J. 1973, 133, 67 Perrin, D. D. in 'Dissociation Constants of Organic Bases in Aqueous Solution', Butterworths, London, 1965 Thunus, L. and Lapiere, C. L. Ann. Pharm. Franc. 1984,42(1),43 Wilson, J. M., Bayer, R. J. and Hupe, D. J. J. Am. Chem. Soc. 1977, 99, 7922 Whitesides,G. M., Lilbum, J. E. and Szajewski, R. P. J. Org. Chem. 1977, 42, 335 Freter, R., Pohl, E., Wilson, J. M. and Hupe, D. J. J. Org. Chem. 1979, 44, 1771 Szajewski,R. P. and Whitesides, G. M. J. Am. Chem. Soc. 1980, 102, 2011 Hansch, C. and Clayton, J. M. J. Pharm. Sci. 1973, 62(1), 1

Int. J. Biol. M a c r o m o l . , 1988, Vol. 10, August

247