The enthalpy titration of troponin C with calcium

342

Biochimica et Biophysica Acta, 535 ( 1 9 7 8 ) 3 4 2 - - 3 4 7 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press

BBA 37967

THE ENTHALPY TITRATION OF TROPONIN C WITH CALCIUM

KAZUHIRO YAMADA *

Department of Physiology, Juntendo University School of Medicine, Hongo, Tokyo 113 (Japan) (Received J a n u a r y 6 t h , 1 9 7 8 )

Summary Microcalorimetric titrations have been used to study the binding of Ca 2÷ to troponin C, the Ca-binding c o m p o n e n t of troponin. Troponin C was extracted from rabbit skeletal muscle and Ca 2÷ was added to Ca-free troponin C in the presence of 1 mM Mg 2÷ at pH 8.83 at 10°C. In these conditions proton exchanges of troponin C on Ca-binding are negligible. In case of troponin C there is neither aggregation nor dissociation when Ca ~+ is added, a phenomenon controlled by bound Ca, which caused some difficulty in the analysis of results for troponin. Using an iterative curve fitting procedure the following parameters of two classes of Ca-binding sites have been determined from the calorimetric results: number of binding sites in 1st class, 1.81; in 2nd class, 0.69; log of binding constant in 1st class, 7; in 2nd class, 5.0; standard enthalpy change for 1st class, --10.00 k J - m o l - 1 ; that in 2nd class, --25.64. The standard free energy and entropy changes have been calculated from these values. The results indicate that the standard entropy change for Ca2+-troponin C interaction is positive and strikingly resembles the values for the interaction of organic sequestering agents such as glycol-EDTA with Ca 2+.

Introduction

Recently Yamada et al. [1] measured the heat of binding of Ca 2÷ to troponin calorimetrically, the magnitude of which is large enough to explain the activation heat produced rapidly on stimulating muscle [2]. Troponin is a complex of three subunits, of which only troponin C binds a significant amount of Ca 2÷ in the physiological range of Ca 2+ concentration [3,4]. The purpose of the present study is to determine the part of this enthalpy change

Abbreviation: G E D T A , glycolethylenediaminetetraacetic acid.

* Present address: Department of Physiology. Oita Medical College, Oita 879-55, Japan.

343 which accompanies the binding of Ca 2÷ to troponin C, and is therefore directly associated with the Ca-binding process. Materials and Methods Troponin was extracted from rabbit skeletal muscle [5], and t r o p o n o n C was separated by column chromatography first on SE-Sephadex C-50 and then on DEAE-Sephadex A-25 in the presence of 6 M urea, 1 mM GEDTA being added in the 2nd step where troponin C was retained and thus separated from troponin I. Troponin C thus prepared has only a low level of bound Ca and was titrated w i t h o u t further treatment. Troponin C prepared for the present series of calorimetry had 0.29 mol Ca per mol protein, measured by atomic absorption spectrophotometry in the presence of 0.4 mM LaC13. All the points in this titration were obtained with the same sample of troponin C. The solutions for the titration were all prepared at one time and stored at 10°C. The buffer solution used contained 0.1 M KC1, 1 mM MgC12 and 25 mM Tris-HC1, pH 8.83 at 10°C. The proton exchanges of troponin C on Ca 2÷ binding are reduced to a negligible level in these conditions. Calorimetric titrations were made with a modified Rhesca conduction microcalorimeter [ 1] at 10°C. After introducing the solutions into the glass calorimeter cells, they were left for about 10 h before mixing was started by rotating the calorimeter. After each reaction the solutions were discarded except in a few cases when the solutions were used for determining the baseline by adding buffer solutions w i t h o u t Ca 2÷. The estimated error of" measurements was 1 to 5%. The whole titration (10 measurements including the baseline determination) was accomplished within 5 days after the solutions were prepared. The total volume for one measurement was 5 ml, 4 ml of protein solution plus 1 ml of Ca 2÷ solution. The reference cell contained the same buffer solution. The sedimentation diagram was obtained with a Hitachi ultracentrifuge. The pH titration was performed using a combination electrode and an air-tight glass cell to replace any CO2 with N2. Results and Discussion The sedimentation diagram obtained with an ultracentrifuge shows that troponin C remains as single molecules irrespective of the level of Ca bound (see also Murray and Kay [6]), whereas troponin aggregates when the level of bound Ca is reduced [1,7]. When troponin C associates with Ca 2÷ at neutral pH, the pH of the solution falls, indicating t h a t protons are released. This is in contrast to the slight alkalinization seen when Ca 2÷ binds to troponin [1]. The titrations were performed at the unphysiological pH of 8.83, because the proton exchanges of troponin C on Ca 2÷ binding are reduced to a negligible level at pH 9 in the presence of 1 mM Mg 2÷. The results were only slightly affected by this high pH condition. The calorimetric titration data are listed in Table I, and are plotted as heat produced against total Ca 2÷ added, both of which are normalized by the a m o u n t of troponin C, in Fig. 1. The enthalpy changes per mol of added Ca 2÷ are much smaller than those previously found for troponin [1]. This might

344 50

.C

,~ 30 Q.

"6 E

10

I

0

I

I

I

I

2 3 4 5 C a / r n o i protein Fig. 1. E n t h a l p y t i t r a t i o n o f t r o p o n i n C w i t h Ca in t h e p r e s e n c e of 1 m M Mg 2+ a t p H 8.83 a t I O ° C (values are given in T a b l e I). A m o u n t of h e a t p r o d u c e d p e r m o l t r o p o n i n C are p l o t t e d against Ca a d d e d p e r tool p r o t e i n . 0 . 2 9 m o l Ca p e r tool p r o t e i n w a s a l r e a d y b o u n d t o t r o p o n i n C b e f o r e t h e t i t r a t i o n s t a r t e d . 1

mean that the enthalpy change accompanying the binding of Ca 2÷ to troponin C is different when the troponin C is in whole troponin. It could also be that when Ca 2+ binds to whole troponin part of the enthalpy comes from changes in the association between the subunits of troponin. However, the larger amount of heat seen with whole troponin could also arise if the dissociation of troponin due to adding Ca 2+ is exothermic. For the binding of Ca 2÷ to two classes of independent sites with the intrinsic binding constants kl and k2 for each of nl and n2 sites in the 1st and 2nd classes respectively, we have [nlkl[Ca] /~/-/1 n2k2[Ca] AH2] Q = Q' + V. [ T N C ] t o t [ ~ + ~ [ C ~ + 1 +~-2[c-a] /

(1)

TABLE I E N T H A L P Y T I T R A T I O N O F T R O P O N I N C W I T H Ca 2+ R e s u l t s of e n t h a l p y t i t r a t i o n of t r o p o n i n C w i t h Ca. V ( t o t a l v o l u m e ) , 5 m l ; [ T N C ] t o t ( t o t a l c o n c e n t r a t i o n of t r o p o n i n C), 8 8 . 2 3 ~tM; p H , 8 . 8 3 ; t e m p e r a t u r e , 1 0 ° C ; [ C a ] , t h e free Ca 2+ c o n c e n t r a t i o n a t equilibr i u m , a n d Qc, t h e " b e s t " f i t t e d v a l u e f o r t h e o b s e r v e d h e a t , w e r e c a l c u l a t e d u s i n g " b e s t " v a l u e s f o r n 1 , n 2 , k I , k 2, A H I , A H 2 ( T a b l e I I ) a n d Q'. Q' ( s y s t e m a t i c e r r o r in d e t e r m i n i n g Q), - - 4 . 4 4 7 m J ; U ( e r r o r s q u a r e s u m ) , 1 . 0 0 4 • 10 -6 j 2 . Q' is larger t h a n e x p e c t e d , since a s e c o n d a d d i t i o n of " z e r o " Ca 2+ s o l u t i o n , to t h e p r o t e i n s o l u t i o n t o w h i c h Ca 2+ h a d b e e n a l r e a d y a d d e d , p r o d u c e d o n l y 0 . 5 5 m J . E i t h e r t h e first a d d i t i o n of " z e r o " Ca 2+ s o l u t i o n w o u l d h a v e p r o d u c e d m o r e h e a t o r s o m e o t h e r c o m p l e x exists. [Ca]tot (#M)

[Ca] (~M)

--Q ( m J)

--Qc ( m J)

62.5 83.3 125.0 125.0 166.6 250.0 333.3 500.0

0.1204 0.2058 0.9433 0.9433 7.808 63.53 142.4 307.0

7.211 9.418 10.60 11.22 14.60 17.98 18.24 18.85

7.644 8.721 11.08 11.08 14.50 17.90 18.46 18.73

345

where Q is the observed heat, Q' a systematic error in determining Q calorimetricaUy affecting each observation equally, V the total volume, [TNC]tot the concentration of troponin C. [Ca] denotes the free Ca:+ concentration at equilibrium and can be determined by solving Eqn. 2 below, stating that the total Ca concentration is the sum of the free and the bound Ca concentrations. T [ r/lkl [Ca ] n:k: [Ca] [Cairo t = [Ca] + [ NC]tot[ l+kl-[C-ai + /

(2)

Employing an iterative procedure [8,9,10] we have found the values of k~, k2, nx, n2, AHI, AH2 and Q', that minimize the value U below, where Qc denotes the right hand side of Eqn. 1, m being the number of observations. rn

u=

j=l

(3)

(Q-Qe)

This is illustrated by the maps (Fig. 2) of U against nl, n: and against log kl, log k2. In the actual analysis an allowance was made for the Ca bound to troponin C (0.29 mol per mol protein) before titrating with Ca 2+. The section marked D represents the "D-boundary" of Sill~n [8], from which the "standard deviation" can be estimated. From Fig. 2a, the "best" values (+S.D.) for nl and n2 are 1.81 + 0.31 and 0.69 -+ 0.48 respectively. The expected values of nl and n2 are 2. Thus nl had about the expected value but n2 is significantly less than that. Fig. 2b shows that the value of log kl that gives Umin is large and its

2.3

15

2.1

13

1.9

11

1.7

9

1.5

7

~D

c

I-3 0

I

I

I

l

I

0.2

0.4

06

08

1.0

n2

1.2

5

I g tog k 2

F i g . 2. ( a ) D e p e n d e n c e o f U o n n 1 a n d n 2. T h e o p e n c i r c l e i n t h e c e n t e r o f t h e " p i t " s h o w s t h e v a l u e s o f n I and n 2 that minimize U (Umi n = 0.89356 • 10 -6 j2). The section marked D is the "D-boundary", w h e r e U - U m i n = Umin/(n - - N ) , n b e i n g t h e n u m b e r o f o b s e r v a t i o n s , N t h e n u m b e r o f u n k n o w n s a n d the degree of freedom (n --N) taken as 6. The S.D. has been estimated as the extreme values of n I and n 2 at the right hand side of this boundary. The circle next to the central open circle represents the section where U = 0.9 • 10 -6 j2 and the outermost incomplete circle U = 1.3 • 10 -6 j2. (b) Dependence of U on logk I and logk 2 . The open circle shows Umin, whereas the solid circle is taken as the "best" point (see text). The vertical line on the open circle represents the section where U equals 0.8936 • 10 -6 j2, almost i n d i s t i n g u i s h a b l e f r o m U m in . T h e s e c t i o n m a r k e d D a n d t h e o u t e r m o s t o n e r e p r e s e n t t h e s a m e v a l u e s o f U as in (a).

346 TABLE

II

THERMODYNAMIC

PARAMETERS

FOR THE BINDING

O F C a 2+ T O T R O P O N I N

C

T h e ' b e s t ' v a l u e s o f n , log/% A H ° a n d the c a l c u l a t e d v a l u e s o f A G ° a n d A S ° a t 1 0 ° C f o r t h e binding o f C a 2÷ t o t r o p o n i n C i n 2 c l a s s e s o f s i t e s . F o r c o m p a r i s o n , v a l u e s o f n a n d l o g k f r o m o t h e r w o r k [ 1 1 ] are also given. Class

n

logh

AH ° (kJ • mo1-1)

AG ° ( k J • rno1-1)

AS ° ( J • d e g - 1 • t o o l -1)

n other work [11]

logk other work [11]

1 2

1.81 0.69

7 5.0

--10.00 --25.64

--37.95 --27.11

98.7 5.2

1.S 2.1

6.5 5.0

" D - b o u n d a r y " wide. This is expected as the value of kl is known to be high and of the order of 104--107 M -1 [11]. Here, the lowest value within its "D-boundary", i.e. 7, was taken as the " b e s t " value for log kl. The " b e s t " value for log k2 can be more accurately determined as 5 + 0.35. The " b e s t " values of nl, n2, k~, k2, AH1 and AH2, together with the corresponding AG ° and AS ° calculated from these are listed in Table II. The association constants k l and k2 thus determined are reasonably close to the values attained by other methods [11]. The Ca2÷-troponin C interaction is thus characterized as having a large positive entropy change, the magnitude of which is strikingly similar to those for the association of Ca 2÷ by organic sequestering agents such as EDTA [12], AS ° being 129.8 J . d e g -1 . m o l -l, GEDTA [13], AS ° 97.1, and diaminocyclohexanetetraacetic acid [14], AS ° 198.8. In two measurements, when Mg 2÷ was added to Ca- and Mg-free troponin C almost no heat change was observed. It is known [11] that troponin C binds Mg 2+ with the association constant of the order of 103 M -~, the standard free energy change being around --16 kJ • mol -~. It follows that the binding of Mg 2÷ to troponin C is entirely entropic, and this also coincides with the case of the organic sequestering agents in general when they associate Mg 2÷ [12--15]. The entropy decrease accompanying the formation of the chelating ring has been estimated [16] as 58.6 J " deg -~" mo1-1 whereas the overall standard entropy change is positive when organic sequestering agents bind divalent metal cations. This positive entropy change has been attributed to the release of water molecules accompanying the complex formation [12--15]. An increase in helical content and other changes indicating more rigid structure of troponin C on binding Ca 2÷ and also on binding Mg 2+ have been reported [17,18]. A striking similarity could thus be infered for the changes of thermodynamic parameters between troponin C and organic sequestering agents when they bind divalent metal ions. A substantial change in ACp is also expected in troponin C solution on binding Ca 2+ . After having completed the manuscript m y attention was drawn to the report by Potter et al. [19]. The main conclusion of the present study is similar to that of Potter et al., although there are substantial differences especially in AH for the 1st class sites (Ca-Mg sites of Potter et al.) as well as in the number of the 2nd class sites (Ca-specific sites of Potter et al.). The AH value for the 1st class sites given b y Potter et al. is substantially more negative than that determined in the present study. Some of these differences could be attributed

347 to the fact that in the present work Mg 2+ is present throughout while Potter et al. measured Ca 2÷ binding to metal-free protein. Although the calorimetric results in the absence of Mg 2+ are not much different from those in the presence of Mg 2+, no a t t e m p t has been made to analyse the observations in the absence of Mg 2+ because of the release of protons. If Mg 2+ and Ca 2+ compete for the same site, the AH values of Ca 2+ binding in the present study would represent the difference between the AH for Ca 2+ and that for Mg 2÷ binding, though the latter is small. It may also be that Mg 2+ and Ca 2÷ binding sites are different. The difference in pH can n o t explain the rather large difference in the AH value for the 1st class sites between the present study and the work b y Potter et al. Since the figures in Potter et al. are for 25°C the difference in AH might thus only be explained by large negative ACp associated with the Ca 2+ binding of troponin C. Acknowledgements It is a pleasure to thank Prof. H. Mashima for his encouragement, Prof. S. Ebashi for his generous guidance in the field of proteins and Dr. R.C. Woledge for helpful discussion. Thanks are also due to Mrs. M. Watanabe for her assistance and to Mr. K. Kabasawa for his aid in computer calculation. This work was supported in part by grants from the Ministry of Education, Japan. The final form of the manuscript was completed while I was staying at the Department of Physiology, University College London, supported by the joint program between the Royal Society and the Japan Society for the Promotion of Science. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Yamada, K., Mashima, H. and Ebashi, S. (1976) Proc. Japan Acad. 52, 252--255 Woledge, R.C. (1971) Pzog. Biophys. Mol. Biol. 22, 37--74 Hartshorne, D.J. and Mueller, H. (1968) Biochem. Biophys. Res. C ommun. 3 1 , 6 4 7 - - 6 5 3 Ebashi, S. (1972) J. Biochem. 72, 767--790 Ebashi, S., Wakabayashi, T. and Ebashi, F. (1971) J. Biochem. 6 9 , 4 4 1 - - 4 4 5 Murray, A.C. and Kay, C.M. (1972) Biochem. 11, 2622--2627 Wakabayashi, T. and Ebashi, S. (1968) J. Biochem. 6 4 , 7 3 1 - - 7 3 2 Sill~n, L.G, (1962) Acta Chem. Scand. 1 6 , 1 5 9 - - 1 7 2 Paoletti, P., Vacca, A. and Arenaxe, D. (1966) J. Phys. Chem. 70, 193--196 I zatt, R.M., Eatough, D., Snow, R.L. and Christensen, J.J. (1968) J. Phys. Chem. 72, 1208--1213 Potter, J.D. and Gergely, J. (1975) J. Biol. Chem. 250, 4 6 2 8 - - 4 6 3 3 Charles, R.G. (1954) J. Am. Chem. Soc. 76, 5 8 5 4 - - 5 8 5 8 Boyd, S., Bryson, A., NancoUas, G.H. and Torrance, K. (1965) J. Chem. Soc. 7353--7358 Anderegg, G. (1963) Helv. Chim. Aeta 46, 1 8 3 3 - - 1 8 4 2 Caxe, R.A. and Stavely, L.A.K. (1956) J. Chem. Soe. 4571--4579 Cobble, J.W. (1953) J. Chem. Phys. 21, 1451--1456 van Eerd, J.P. and Kawasaki, Y. (1972) Biochem. Biophys. Res. C ommun. 4 7 , 8 5 9 - - 8 6 5 Kawasaki, Y. and van Eerd, J.P. (1972) Biochem. Biophys. Res. C o m m u n . 4 9 , 6 9 8 - - 9 0 5 Potter, J.D. Hsu, F.-J. and Pownall, H.J. (1977) J. Biol. Chem. 252, 2 4 5 2 - - 2 4 5 4