Kinetic correlations between the disulfide bond reduction and the induced conformational change of proteins

89

Biochimica et Biophysica Acta, 668 (1981) 89--97 © Elsevier/North-Holland Biomedical Press

BBA 38631

KINETIC C O R R E L A T I O N S BETWEEN T H E DISULFIDE BOND REDUCTION AND T H E INDUCED C O N F O R M A T I O N A L CHANGE OF PROTEINS

TATSUHISA SEGAWA, KUNIHIRO KUWAJIMA and SHINTARO SUGAI

Department of Polymer Science, Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060 (Japan) (Received September 15th, 1980)

Key words: Disulfide bond reduction; Dithioerythritol; Conformation change induction; ~-Lactalbumin; Trypsin inhibitor; Circular dichroism

Summary Kinetic correlations between t he disulfide bond reduct i on in excess dithioerythritol and th e induced c onf or m at i onal change were studied on two proteins, bovine ~-lactalbumin and soybean trypsin inhibitor, at 25°C and pH 8.0-8.5 b y measuring the absorbance o f oxidized d i t h i o e r y t h r i t o l at 310 nm and t h e ellipticity at 270 nm, respectively. With ~-lactalbumin, in t he absence o f guanidine h y d r o c h l o r i d e (Gdn • HC1) or in dilute Gdn • HCI, the kinetics for t he bond reduct i on and t he conformational change were b o t h o f a biphasic t ype. The fast phase was com pl et e within a few seconds and was associated with t he reduct i on of some o f the disulfide bonds and with almost com pl e t e loss o f t he tertiary structure. The slow phase was associated with t he r educt i on o f ot he r disulfide bonds. In concent rat ed Gdn • HC1, the kinetics of b o t h processes were observed as a single phase, t he rate o f which was similar to t ha t o f the slow phase in t he absence o f Gdn • HC1 or in dilute Gdn • HC1. In all cases studied, the rate o f the bond reduct i on was similar to t h a t o f t he conf or m a t i ona l change induced. By correcting t he change in absorbance at 310 nm to a c o n t r i b u t i o n from the protein due to the conformational change, t he n u m b e r o f bonds which are reduced in t he fast phase in t h e absence o f Gdn • HC1 was determined to be 1.0--1.1. It was shown, taking observations o f others and theoretical results into account, that the bond reuced in th e fast phase might be t he one bet w een Cys 6 and Cys 120. On the o t h e r hand, one of two bonds o f soybean trypsin inhibitor in the native form was reduced in the fast phase w i t h o u t any loss o f the tertiary structure, and th e o t h e r was reduced in t he slow phase. Considering t he results o f o t h e r researchers, it w a s concluded t hat t he bond reduced in t he fast phase of t h e inhibitor is a 136--145 bond.

90 Introduction The disulfide bonds of a protein play a key role in the stabilization of its native three
91 Materials and Methods

Materials. Bovine a-lactalbumin was prepared from fresh milk b y the m e t h o d previously described [ 13]. Recent studies in our laboratory have indicated that bovine, human and goat a-lactalbumins prepared b y the ordinary method include one calcium ion per molecule [14]. The calcium ion plays an important role in the maintainance of the native structure of a-lactalbumin. The calcium ion has not been eliminated from the a-lactalbumin used in this study. Soybean trypsin inhibitor was purchased from Worthington Biochemical Co., and it was purified b y chromatography on DEAE-cellulose (DE 32) according to the method of Yamamoto et al. [15]. Disc gel electrophoresis showed that the proteins are monodisperse. Protein concentrations were determined from absorption measurements at 280 nm using J-~lcm~l%values of 20.1 (a-lactalbumin) [16] and 10.1 (soybean trypsin inhibitor) [15]. Gdn • HC1, dithioerythritol and 5,5'dithiobis(2-nitrobenzoic acid) were obtained from Nakarai Chem. Ltd., Kyoto, as the specially prepared reagent grade and they were used without further purification. The concentration of Gdn • HC1 was estimated b y refractive index measurement at 589 n m [ 17 ]. The concentration of dithioerythritol was determined b y the method of Ellman [18]. Other chemicals used were of reagent grade from Nakarai Chem. Ltd. Methods. Equilibrium CD measurements were taken on a Jasco J-20 spectropolarimeter, using a cell o f 1 or 10 mm pathlength. The disulfide bond reduction was carried out in the presence of a 20-fold excess of dithioerythritol over the disulfides in the proteins. The rates of bond reduction in the proteins were measured b y a Union RA 1100 stopped-flow spectrophotometer [19,20] or a Union SM 401 spectrophotometer with a mixing apparatus [21]. The fast CD measurements were done by the method described previously [ 11 ] using a Union CD 1000 stopped-flow spectropolarimeter and a cell of 10 mm pathlength. All procedures were performed under nitrogen gas at 25 -+ 0.1°C. Because dithioerythritol forms an intramolecular cyclic disulfide bond and has a relatively low redox potential, the thiol
Ai exp(--kit)

(1)

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where A(t) is an observed change in absorbance at time t, ki the i-th apparent rate constant and Ai the i-th kinetic amplitude. The kinetic parameters, Ai, A® and ki, were calculated b y the non-linear least squares method (Gauss-Newt o n method ) to get the best-fitting curve. The kinetic parameters associated with the conformational change were also calculated b y the same method. The numbers of free thiol groups in the reduced proteins were determined b y Ellman's m e t h o d with 5,5'~lithiobis(2-nitrobenzoic acid) after separation of the proteins from dithioerythritol on a Sephadex G-25 column equilibrated with 10 mM HC1.

92 Results a-Lactalbumin Fig. 1 shows the CD spectra of a-lactalbumin in four conditions at pH 8.0: the native form in the absence of EDTA, the disulfide intact form in the presence of EDTA, the intact form in the presence of EDTA and 4 M Gdn • HC1, and the completely reduced form, in which the number of the thiol groups was found to be 7.3--7.5. The tertiary structure measured by the aromatic CD spectrum is almost completely disrupted by the reduction of disulfide bonds, although the backbone secondary structure measured by the far ultraviolet CD spectrum is partially retained even in the completely reduced form. The kinetics of disulfide bond reduction by dithioerythritol is shown in Fig. 2. In the absence of Gdn • HC1, the kinetics is found to be biphasic: the fast phase within a few seconds associated with the reduction of some of the disulfide bonds and the slow phase with the reduction of the other bonds by the reagent. At 4 M Gdn • HC1, on the other hand, the reaction was observed as a single phase. Thus, all of the disulfide bonds are reduced at the same rate for the molecule unfolded by Gdn • HC1, and the rate is comparable to that of the slow phase in the absence of Gdn • HC1. The kinetics of the conformational change induced by the disulfide bond reduction is shown in Fig. 3 as a time-dependent change in ellipticity at 270 nm, [0 ]270. In the absence of Gdn • HC1, the kinetics is also biphasic, and most of the change in [0]270 occurs in the fast phase. Thus, the disruption of the

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Fig. 3. K i n e t i c s o f t h e e o n f o r m a t i o n a l c h a n g e o f ~ - l a c t a l b u m i n i n d u c e d b y t h e d i s u l f i d e b o n d r e d u c t i o n at p H 8 . 0 a n d 2 5 ° C (a) in t h e a b s e n c e o f G d n • HCI; ( b ) in t h e p r e s e n c e o f 1 . 5 M G d n • HCI. T h e c o n c e n t r a t i o n s o f t h e p r o t e i n a n d d i t h i o e r y t h r i t o l in a q u e o u s 1 m M E D T A s o l u t i o n s u s e d w e r e t h e s a m e as i n Fig. 2.

disulfide bonds reduced in the fast phase leads to a remarkable loss of the tertiary structure of the protein. In concentrated Gdn • HC1, the time course of the change in [0]270 was also observed as a single phase. There is a relatively small conformational change caused by the disulfide bond disruption even after the protein has been unfolded by G d n . Hcl (Fig. 1, curves c and d). At 1.5 M Gdn • HC1, the ellipticity change occurs in the time range corresponding to that in Fig. 2b, and the kinetic amplitude is small as compared with that in the absence of Gdn • HC1, The parameters o f the bond reduction and the conformational change were calculated from the kinetics as shown in Figs. 2 and 3 according to Eqn. 1. The best-fitting curves are obtained by assuming n = 2 in the absence of Gdn • HC1 or in the dilute G d n . HC1 (<1.5 M). The apparent relaxation times of both reactions, bond reduction and conformational change, are shown in Fig. 4, in

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Fig. 5. C D spectra o f s o y b e a n trypsin inhibitor in a q u e o u s Tris-HC1 o f 5 0 m M ( p H 8 . 5 ) w i t h 1 m M E D T A at 2 5 ° C , (a) in t h e i n t a c t f o r m ; (b) in the c o m p l e t e l y r e d u c e d f o r m (after r e d u c t i o n for 1 h b y d i t h i o e r y thritol). Fig. 6. Kinetics, (a) o f the disulfide b o n d r e d u c t i o n b y d i t h i o e r y t h r i t o l ; (b) of the i n d u c e d c o n f o r m a tional c h a n g e , o f s o y b e a n trypsin i n h i b i t o r in a q u e o u s Tris-HCl o f 5 0 m M ( p H 8 . 5 ) a n d 1 m M E D T A at 2 5 ° C . T h e a q u e o u s s o l u t i o n o f the p r o t e i n o f 8 4 p M and t h a t o f d i t h i o e r y t h r i t o l o f 3 4 m M w e r e m i x e d in the s t o p p e d - f l o w apparatus.

which the relaxation time of the bond reduction is found to be similar to, but a little smaller than, that of the conformational change in both the fast and slow phases.

Soybean trypsin inhibitor The CD spectra of the inhibitor in the native and reduced forms are shown in Fig. 5. The spectrum in the near ultraviolet region shows a large negative band centered at 273 nm as observed for a-lactalbumin, but in the far ultraviolet region a large positive band at 226 nm is found. The latter may be due to tyrosine residues, because soybean trypsin inhibitor is known to have little backbone secondary structure [24,25]. The reduction of two disulfides disrupts the tertiary structure in the protein almost completely. In Fig. 6a, the rate of disulfide bond reduction is shown. The reduction is apparently composed of two phase and the bond reactivity of the inhibitor is more sluggish than that of a-lactalbumin. The kinetic curve of the conformational change induced by the bond reduction is also expressed in terms of [0 ]27o in Fig. 6b, where only the slow phase corresponding to the slow phase in Fig. 6a is seen. The fast reduction of disulfide bond of the inhibitor does not induce any conformational change. The apparent relaxation times were calculated from Fig. 6a and b. The relaxation time in the fast phase from absorbance at 310 nm was 180 s and that

95 in the slow phase 2100 s. The relaxation time in the slow phase from [0]27o was 2300 s, which agrees well with that from absorbance. Discussion Iyer and Klee [9] demonstrated that the change in absorbance at 310 nm is parallel with the change in the extent of the reduction of the disulfides in a protein, because most proteins have only negligible absorbance at 310 nm. However, it has also been known that the unfolding of some proteins which contain t r y p t o p h a n brings about an unusual difference spectrum at a wavelength longer than 300 nm [26,27]. a-Lactalbumin shows a positive difference absorption band at 302--303 nm when unfolded by acid, heat or Gdn • HC1 [28]. The acid transition of the protein, which leads to a partial unfolding of the molecule as brought about by the disulfide bond reduction, is accompanied with a change in molar extinction coefficient at 310 nm of 130 M - t . cm -1 [28]. This change in extinction coefficient is not negligible in view of the change corresponding to the reduction of a disulfide brand, 110 M -t • cm -~ [9]. Thus, in order to estimate the number of disulfide bonds reduced in the fast phase of the reduction of a-lactalbumin, the absorbance change induced by the unfolding should be taken into account. The existence o f EDTA for elimination of heavy metal ions should also be taken into consideration. EDTA extracts a calcium ion from holo~-lactalbumin and it is known to destabilize the tertiary structure even in the intact a-lactalbumin [14]. The disulfide bonds in the unfolded intact a-lactalbumin are reduced in the slow phase. The fraction fu of such unfolded intact a-lactalbumin in aqueous 1 mM EDTA at pH 8.0 may be estimated from the following:

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where [0 ]270 is the observed ellipticity in the presence of 1 mM EDTA, [0 ]No the ellipticity of the native intact a-lactalbumin in the absence of EDTA and [0]vT0 that of the unfolded intact form. Because [0]ST0 =--300, [012~70 = - - 3 0 and [0]27o = - - 1 7 0 deg • cm 2 • dmo1-1 as shown in Fig. 1, fv = 0.48 in aqueous 1 mM EDTA at pH 8.0 and 25°C. As a first approximation, the number n~ of the reduced disulfide bonds per molecule in the fast phase in the absence of Gdn • HC1 is related to the kinetic amplitide, Ae kin, in the fast phase calculated from the time course in Fig. 2a as follows: A 6 kin = ( 1 - -

fv)(nf Ae s - s + A6~ ° n f )

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where Ae ~s is the change in molar extinction coefficient of oxidized dithioerythritol at 310 nm and Ae~°nf is that due to the conformational change in the fast phase at 310 nm. Here, we assume that Ae~T M is equal to Ae at 310 nm caused by the native to acid transition of a-lactalbumin [13,20,28], because the tertiary structure is almost completely disrupted in the fast phase although the backbone secondary structure is considerably conserved, as shown in the acid form. The kinetic amplitude in the slow phase, Aeski~ , is also expressed with the number of nt as follows:

96 Ae~ TM = (4 - - n f ) AeS-S(1 - - f v ) + 4 AeS-Sfc

(4)

where the change in molar extinction coefficient at 310 nm due to the conformational change is assumed to be negligible in the slow phase. By using Ae s-s = I I 0 , Ae~ °nf = 130, /~e~TM = 129 and Ae~ TM = 388 M -I • cm -~, n~ is found to be 1.0--1.1 from Eqns. 3 and 4. Therefore, it is concluded that one of the four disulfide bonds in a-lactalbumin is reduced in the fast phase. Studies by Shechter et al. [3] showed that a disulfide bond between Cys 6 and Cys 120 in bovine a-lactalbumin is more susceptible to reduction by dithioerythritol than other bonds. Also, according to the molecular model of the protein by Warme et al. [29]. the bond 6-120 seems to be exposed on the molecular surface and the bond may be in significant conformational strain. Thus, the bond is easily reduced by dithioerythritol and the rate of reduction is faster than that of the model disulfides such as glutathione [23]. The bond reduced in the fast phase in this work may be the 6-120 bond. The 6-120 bond reduction is accompanied by disruption of the tertiary structure under the present conditions. Iyer and Klee [9] have found that at low temperature (approx. I°C) only one of the four disulfide bonds of a-lactalbumin is reduced by dithiothreitol. Our recent studies have also shown that at low temperature one of the disulfide bonds of the protein is reduced w i t h o u t loss of the tertiary structure and that the three disulfide a-lactalbumin is unfolded remarkably at 25°C because it has relatively low stability against thermal unfolding [30]. a-Lactalbumin with two carboxymethylcysteines at position 6 and 120 is reported to be able to bind anti~-lactalbumin antibodies at 4°C, but to have only about 50% of the activity of the native protein in the lactose synthesis reaction and about 35% of the inhibitory capacity in the inhibition of N-acetyllactosamine synthesis [ 3]. G d n . HC1 affects not only the conformation of a-lactalbumin, but the relaxation time of bond reduction. Creighton [31] observed such dependence of relaxation time of the bond reduction of pancreatic trypsin inhibitor on G d n " HC1 concentration. Also, he showed a linear relationship between the rate of reduction and the ionization t e n d e n c y of the thiol groups generated by the reduction of several model disulfides. Aqueous Gdn • HC1 may affect the apparent pK value of the thiol group generated in a-lactalbumin. On the other hand, one of the disulfide bonds in soybean trypsin inhibitor has been indicated to be reduced by treatment with 0.25 M sodium borohydrate at 0°C without loss of activity [2]. The bond position was determined after carboxymethylation of the reduced protein and it is considered to be the 136--145 bond [32]. The present study indicated that the disruption of the 136--145 bond does not lead to any change in conformation. X-ray analysis [25] reveals that the bond is exposed on the surface of the molecule. Its disruption does not lead to a change in conformation. This bond is not essential for maintenance of the native conformation. The other bond, 39--86, is reduced very slowly, and its reduction is accompanied by the complete disappearance of its tertiary structure with loss of the inhibitory activity. The bond 39--86 seems to be buried in the interior of the molecule. X-ray results are consistent with such a conclusion. The kinetic correlation studies between the disulfide bond reduction and the

97 conformational change induced will be important to make clear the roles of the disulfide bonds for conservation of the native structure and for the folding process of protein. Acknowledgements The authors wish to thank Ministry of Education of Japan for financial support, and Professor Michio Yoneyama for his advice.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

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