Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride

ELSEVIER Biochimica et Biophysica Acta 1207 (1994) 223-230

Biochi~ic~a et Biophysica A~ta

Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride

Zulfiqar Ahmad a,,, Faizan Ahmad b

a Centre for Biosciences, Jamia Millia Islamia, New Delhi-110025, India b Department of Chemistry, Jamia Millia Islamia, New Delhi-110025, India

Received 5 October 1993; revised 7 March 1994

Abstract

Cytochrome c (cyt c) denaturation by calcium chloride (CaC12) and guanidine hydrochloride (GdnHC1) denaturation in presence of low fixed concentrations of CaC12 has been carried out by UV/Vis spectrophotometry at pH 7.0 and 25°C. The unfolding process was followed by measuring changes in difference molar extinction coefficient around 400 nm and de29 o. The products of denaturation were further characterized by intrinsic viscosity ([7/]) measurements. It has been observed that the reversible unfolding of cyt c by CaC1 z occurs in two distinct stages or involves three species (or states) namely N ~ X ~ D. Characterization of the native state, N intermediate state, X and the end product, D suggests that (i) During N ~ X only heme is exposed and no secondary structure unfolding occurs so that X state remains as compact as the native state. (ii) Stage X ~ D shows the melting of secondary structure. (iii) The end product corresponds to a random coil. and (iv) Thermodynamic characterization of the end product shows that heme plays an important role in the stability of the protein and removal of heme will lead to the unfolding of cyt c. Mixed denaturation shows a highly cooperative reversible transition between the native and denatured conformation. Analysis of the mixed results shows that (1) Gdn ÷ does not have any binding site (s) on the native cyt c, (2) there is one binding site for Ca 2÷ which stabilizes the protein, and (3) the binding constant, ks, is 5 M- for Ca 2+.

Key words: Denaturation; Cytochrome c; Guanidine; Calcium chloride

I. Introduction

The folding unfolding processes can lead to a better understanding of the structural, conformational and possi- bly functional aspects of proteins and enzymes. The unfolded or denatured state under physiological conditions D O is the most relevant to an understanding of protein stability, since stability can be defined as

AG H2° = - RT In [D O ] / [ N ] (1)

It is known that most proteins under native conditions, i.e., in water or dilute buffer, are stable by 5 kcal to 20 kcal mo1-1, so only one out of every 104 to 1015 protein molecules is in the D O state. Consequently, experimental studies have concentrated on D state under conditions quite different than aqueous buffer at physiological pH near 25°C. By analyzing how the properties of denatured

* Corresponding author.

0167-4838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4838(94)00045-1

states vary under these more extreme conditions, estimates of the properties of D O can often be made by extrapolation back to conditions where N, the native state, exists [1].

Earlier studies by inorganic salts on different proteins [2-12] show that the denatured states obtained by these salts are intermediate states between the native and GdnHC1 or urea denatured states. It has also been shown at least in some cases that residual structures remained in partially denatured states can be removed by addition of urea [6,7].

It means different denaturants give rise to different denatured states of a protein, i.e., conformation with different amounts of secondary and tertiary structure. The ques- tion arises, why do proteins behave like that? Ahmad and Ahmad [13] have suggested that this could be due to the presence of two or more independent substructures of different stabilities that a folded protein may contain. They [13] denatured cytochrome c (cyt c) by GdnHCI and lithium salts and have shown that cyt c, having only one domain [14,15], gives the same completely denatured state in both.

224 Z. Ahmad, F. Ahmad / Biochimica et Biophysica Acta 1207 (1994) 223-230

In this study we are reporting results of CaCI 2 denaturation and GdnHCI induced unfolding in presence of low fixed concentrations of CaCI 2. The former study suggests a possible mechanism of CaC12 denaturation. From the later results it is proposed that cyt c have a binding site for the Ca 2+ which stabilizes the native protein leading to a

H20 higher value for the protein stability (AGap p ).

2. Materials and methods

Horse heart cyt c, type VI was purchased from Sigma, USA. Its purity was rechecked on Sephadex G-75. CaCI 2 and other chemicals obtained from Aldrich, USA were analytical grade reagents. Ultra pure GdnHC1 was obtained from Schwarz/Mann Biotech. All solutions including buffers were made up using doubly distilled water.

Cyt c solution was oxidized by adding 0.1% potassium ferricyanide, and dialysed extensively against several changes of 0.1 M KCI solution (pH 7.0) in cold. Concen- tration of the stock cyt c solution was estimated spec- trophotometrically using a value of 106.1 × 10 3 M - l c m -1 for the molar extinction coefficient at 410 nm [16].

Stock denaturant solutions were prepared in 0.03 M cacodylate buffer containing 0.1 M KC1 (pH 7.0). Concen- tration of stock solutions of CaCI 2 [17] and GdnHC1 [18] were determined by measuring the difference refractive index of each solution and 0.03 cacodylate buffer containing 0.1 M KCI.

All weighings were done on Sartorius Analytical A200S balance and pH measurements were made using Consort P-907 pH-meter.

2.1. Preparation of solutions for bsothermal denaturation experiments

(i) Single denaturant system. For unfolding experiments, known amounts of stock protein solution, buffer and denaturant solution (CaC12) were mixed and incubated overnight which was sufficient for completion of the reaction. A similar procedure as used in unfolding experiments was employed in preparing the solutions for refolding experiments with the only exception that cyt c was first denatured in concentrated denaturant solution and then diluted with the buffer.

(ii) Mixed denaturant system. For the denaturation by mixed denaturants (GdnHC1 and CaC12) samples were prepared in the same way as for single denaturants except that after adding protein solution to a series of flasks varying amounts of buffer, stock GdnHCI solution and a fixed volume of CaCI 2 solution was added to the flasks. All the solutions were incubated overnight. Refolding was observed by denaturing the protein with concentrated solution of both denaturants and then diluting with buffer.

Difference spectroscopic measurements were performed in Shimadzu 2100 UV/Vis spectrophotometer using a

well matched 1-ml quartz cuvetts of I cm path length. The temperature of both cells were maintained at 25 + 0.05°C by circulating water from an external thermostated water- bath (Shimadzu TB-85). Difference spectrum were obtained by subtracting the spectrum of the native protein from the protein exposed to denaturants.

Viscosity measurements were made on an Ostwald type viscometer with a flow rate of 60 s for 1 ml of distilled water. The viscometer was kept in a thermostated water- bath. All measurements were performed at 2 5 _ 0.05°C. The timing was done manually with a stop watch which can be read to 0.1 s. r/red, the reduced viscosity of protein solution was calculated using the following relationship [191.

t - t o 1 - ~2 Po rite a - - + - - (2)

t o c Po

where t o and t are the times of fall of one ml solvent and one ml solution, respectively, c is the protein concentration in g /ml , U2 the partial specific volume of cyt c and Po the density of the solvent. A value of 0.724 m l / g for for U2 was used [20].

2.2. Analysis of the transition curves

The unfolding transition curves of cyt c fall in to two categories which were analyzed as follows: The unfolding induced by GdnHCI in presence of low fixed concentrations of CaC12 is assumed to follow a two-state mechanism, N ~ D. The denatured protein fraction, fD, was calculated using the following equation.

(Y - Y N ) fD (3)

(YD -Yr~)

where y is the observed conformational property and YN and YD represent values of y obtained for the protein in the native and fully denatured states. These values need not be constant and are described as functions of [d], the denaturant concentration.

The state of equilibrium can be represented in terms of an apparent equilibrium constant (Kap p) by the relation

fD (Y --YN)

Kapp (1 - - f o ) (YD--Y) (4)

The apparent free energy change on unfolding was calculated by using the relationship

AGap p = - R T In Kap p (5)

where R is the gas constant and T the temperature in Kelvin.

The CaC12 induced unfolding of cyt c involves two separable stages, namely N ~ X and X ~ D, where X is the intermediate state. It has been assumed that each stage follows a two-state mechanism.

Z. Ahmad, F. Ahmad / Biochimica et Biophysica Acta 1207 (1994) 223-230 225

This follows that the fraction of the intermediate state, #x, in the reaction N ~ X can be obtained from the relation

(Y - Y N ) fx = (6)

(Yx - Y ~ )

where fx + fN = 1. Furthermore the fraction of the denatured species fD in reaction X ~ D can be estimated from the relation

(y-yx) fD ( 7 )

(YD --Yx)

where fx + fD = 1. Consequently the corresponding equilibrium constant and free energy changes for the process N ~ X are

Kx = f x / ( 1 - f x ) , AGx = - R T In K x (8)

and for the process X ~ D

KD - - f D / ( 1 - - fD) , AGo = - R T In K o (9)

For each process be it N ~ D, N ~ - X or X # D, AG values in the range - 1.36 _< AG (kcal mol- 1 ) _~< 1.36 were plotted against the molar concentration of the denaturant, [d]. A least squares analysis was used to fit the data using the relationship shown by the Eq. (10).

AG = m o H2° - m[d] (10)

where AG H2° is the value of AG at zero molar denaturant concentration, and m is the slope of the line, i.e. (aAG/O[d])r, P. The midpoint of transition curve, Cm, was calculated from C m = AGn2°/m.

3. Results and discussion

Cyt c has been denatured by a number of agents and studied by both kinetic and equilibrium approaches, yet there is no consistent view regarding either the mechanism for denaturation or folding of the protein or an understanding of the processes in terms of various structural aspects of the molecule,

Mayer et al. [21,22] and Stellwagen [20] studied the urea denaturation of cyt c and proposed a two-step process, i.e., a three state model. Similar inferences were made when denaturation was induced with a variety of alcohols [23] while a simple single-step process has been proposed by McLendon and Smith [24] and Aviram and Weissmann [25]. It is thus obvious that no clear cut picture emerges from the earlier studies regarding the consistency of either mechanism or free energy change, A~ H2° asso- --~app ,

ciated with different transitions. During the course of this study we have observed some

very interesting results on the denaturational behaviour of this protein which are presented and discussed below.

2 z :E v

7 0 17,

1.C

0.~

Q6

0.4

0.2

O.C

i , i i i

Y x A

.00,.0.,0.0_0,. .0..0~C¢~0~0" ~ ~ Y N

l l l l I

B / fF

f×i ! fD

i

\ \ \

\ \ \

\ \ \ \ \

i i

c

\ \ \ \

o

4

3 v o ~b

2 .~

o

-2

Fig. 1. Changes in Ae405 of cyt c on denaturation by CaC12 at pH 7.0 and 25 + 0.05°C (A) transition curve, (B) normalized transition curves, (.fx): N ~ X, (fD) X ~ D, and (C) variation of AG x and AG D as a function of CaCI 2 concentration.


3.1. Single denaturation system

Fig. 1 shows the isothermal denaturation of cyt c by CaC12 in the visible region. Difference molar extinction coefficient value at 405 nm, at which peak occurs, is the wavelength where the maximum value for difference spec- tra for hemopeptides occurs [26]. This spectral property measures the interaction of heme with the globin chain [13].

It can be seen in Fig. 1A that the spectral property increases linearly with an increase in CaCI 2 concentration from 0-1.50 M. This represents a pretransition region solvent effect on the protein. This can be defined by the relation YN = (1567 _+ 89) × [CaCI2]. The conformational changes appear to start above 1.50 M [CaC12]. This increase in conformational property occurs upto 2.90 M where the value of optical property is 24 000 M - 1 c m - 1, a value observed for cyt c denatured by GdnHC1 and lithium salts in buffer [13].

It is interesting to no te that Ae decreases with an increase in CaC12 concentration above 2.90 M. This change is sigmoidal. It appears that this represents another change in the conformation of the protein. From 4.25 M [CaCI 2] onward the decrease becomes linear (post-transition region). A linear extrapolation of the posttransition region results to 0 M [CaClz] can be described by the relation

YD = (24000 _+ 308) -- (3032 _+ 68) X [CaCI2]. Here again the value of YD is 24000 M-1 cm-1 at 0 M denaturant which is in excellent agreement with those reported earlier for a completely unfolded cyt c [13,27,28]. From these results it appears that CaC12 induced unfolding of cyt c occurs in two stages namely N ~ X , where X is the intermediate state and X ~ D.

Fig. 1B shows the normalized transition curves obtained by plotting fx and fD against [CaC12]. These were calculated according to the Eqs. (6) and (7), respectively. In the determination of fx a value of Yx = 24000 M -1 cm -1 was used. A plot of AG x and AG D as a function of [CaC12] is shown in Fig. 1C.

All thermodynamic parameters obtained from the analysis of the free energy changes versus [CaC12 ] plots according to Eq. (10) are AGx n2°= 8 .31_ 0.18 kcal mol - ] , m = 3.55 + 0.08 kcal /mol mo1-1 and C m = 2.43 M for phase I, i.e., for the process N ~ X. The thermodynamic values for the phase II, i.e., for the process X ~ D are AG D = 1.46___ 0.07 kcal mo1-1, m = 4.05 ___ 0.18 kcal /mol mo1-1 and C m = 3.26 M. AG o represents the value obtained from extrapolation of AG D values upto the starting of the process of X ~ D.

Since AGH~, ° is a thermodynamic function and does not depend on path, it is therefore expected that AGa~ ° asso- ciated with N ~ D transition should be sum of Gibbs

i i i ) i i i i

A C 0 0 0 0 0 0 0 0 - 0 - % -YN

o o '-' _ _o o_ .o__ y x o o o%o~ oc~

,I-

I 1 1 \

f <] % / / \ 5

/ \ 12 / "

\ 4 o ,, E

o? /o :, o \ / \

0.8

r \ ,~o .< 0.~ ) " . 1

Q4 0

\ \ o/ /9 \ -1

, / O.C- o - o - o - - o - o - o J -2

I I I

Fig. 2. Changes in A / ~ 2 9 0 of cyt c on denaturation by CaCI 2 at pH 7.0 and 25 :k 0.05°C (A) transition curve, (B) normalized transition curves, (fx): N ~- X, ( fo ) X ~ D, and (C) variation of AG x and AG O as a function of [CaCl2].


energy changes for N ~ X and X ~ D. Thus we get a value of 9.77 + 0.13 kcalmo1-1 for AG H2° the free en- - - - - app ,

ergy change which is the sum of AG n2° and AG o. CaCl 2 induced denaturation of cyt c was also followed

by observing changes in Ae290, which measures local change in the environment of tryptophan and heme [13] (see Fig. 2A). In the region 0-1.50 M CaC12 no change in optical property occurs (i.e., YN = 0 . 0 ) . Above 1.50 M [CaCI2] A,~ value starts decreasing with an increase in the concentration upto 4.25 M CaC12. There occurs a linear increase in A s values above 4.25 M CaCl 2. Extrapolation of all results from 4.25-6.00 M gave a value of - 13 000 M - l c m -1 for YD which is identical to what has been found for cyt c denatured by GdnHCl and lithium salts in buffer [13].

In contrast to the results shown in Fig. 1A no separable intermediate can be seen in Fig. 2A, however, the transition profile is asymmetric. Since asymmetry can only mean that there are at least two stages in the change in the environment of heme and tryptophan during denaturation, we divided the transition curve into two transitions; namely, N ~ X and X ~ - D . A value of - 4 0 0 0 M - l c m -1 is obtained for Yx. This division was based on our visible region studies.

Mayer et al. [21] have shown that by adopting the simple procedure of generation of a transition profile by peeling off the contribution of a given transition we can effectively resolve the absoptivity-denaturation profile into its constituents.

The values of the fractional change for transition N ~ X and X ~ D were converted into and K x and K D and thus AG x and AG D values using Eqs. (8) and (9), respectively (see Fig. 2C). A least squares analysis of the data according to Eq. (10) yielded the thermodynamic parameters which are as follows: AG n2° and AG D = 6.97 _ 0.09 and 1.66 _+ 0.08 kcal mo1-1, m = 3.04 + 0.04 and 1.88 + 0.09 kca l /mol mo1-1 and C m = 2.29 and 3.78 M for phase I and phase II, respectively. A value of 8.63 kcal mol-1 is observed for AG~H~ ° by adding AG n~° and AG D.

It is interesting to note that f values as well as AG values determined at two different wavelengths fall on the same transition curve for step I but not for step II. The coincidence among the results obtained by two different physical properties suggests that the denaturation of cyt c by GdnHC1 and lithium salts is very close to a two-state transition [13,29]. This appears to justify our analysis of transition curve obtained from observing changes from Aez90. Furthermore our assumption that transition N ~ X and X ~ D represent two two-state transitions seems to be correct. It should, however, be noted that such coincidence or non coincidence can neither proves or disproves for the existence of intermediates on transition between N ~ - D states. Dill and Shortle [1] have provided a theoretical argument for a two-state transition followed by two different conformational properties which do not coincide when these different transitions were normalized.

18

16

14

12

10

f f

w ~ ~ ~- - - - - - -o - - - - - - - - - - - - .o

[ [ I 1 I I 0 4 8 12 16 20

EProtein-~ x 103g/ml

Fig. 3. Reduced viscosi ty of cyt c at p H 7.0 and 2 5 + 0 . 0 5 ° C in ( Q )

buffer, ( 0 ) 2.90 M CaC12, ( A ) 4.25 M CaCl 2 and ( D ) 3.50 M GdnHC1.

A(~rHzO value of 7 + 1 kcal mo1-1 has been reported - app

earlier for cyt c denatured by GdnHCI and lithium salts in buffer [13,24,29]. It can be seen that a higher value of AGH~ ° is obtained for CaC12 denatured cyt c. The best possible explanation could be that there exist binding site(s) for Ca 2+ on the native cyt c which stabilizes the protein leading to a higher value for the conformational stability. This shall be discussed in the section of mixed denaturation system.

The product of CaC12 denaturation were also characterized by [7/], the intrinsic viscosity measurements. We have measured the 7/red of cyt C in 2.90 M CaCI 2 at which cyt c is in the X state and in 4.25 M CaC12 at which protein is in the D state, as a function of protein concentration. Analysis of both sets of data using Eq. (2) gave values of 3.60 + 0.11 and 14.62 _+ 0.13 m l / g for the value of [T/] of X and D states, respectively (see Fig. 3). It can be seen in Fig. 3 that the value of [7/] increases from 2.67 ml /g , obtained for the native protein in buffer to a value of 14.61 _+ 0.91 m l / g for the protein in 3.50 M GdnHC1. It


can also be seen that the value of [~/] in the X and D states are in excellent agreement for the values reported in concentrated solutions of GdnHCI and lithium salts [13,20,27,30]. A comparison of the [7] values of the native and X states shows that the value for X state is slightly higher than that of the native state. This suggests that the gross conformation of the protein does not change much during the intermediate state, i.e., the X state is as compact as the native state or the intermediate state retains all the secondary structure of the polypeptide backbone.

Characterization of the intermediate, X and end product, D by UV/Vis difference spectroscopy and intrinsic viscosity measurements suggests that (i) during phase I, i.e. N ~ X, heme gets exposed to the solvent without any melting of the polypeptide backbone conformation and (ii) in phase II, i.e. X ~ D, the actual unfolding of the secondary structure occurs. Therefore, we can say that CaCI 2 induces a biphasic transition in cyt c and a possible mechanism of denaturation of cyt c by CaCI2 is N ~ X ~ D .

It is interesting to note that Ahmad and Ahmad [13], Ikai et al. [27], Ohgushi and Wada [31] and Goto et al. [32] have also shown the presence of an ordered structure in cyt

c between the process of N ~ D which is as compact as the native protein.

On the basis of thermodynamic results of the process N ~ X ~ D it appears that heme plays the main role in maintaining the three-dimensional structure of cyt c. CaCI 2 induced transition curve for the process N ~ X gave an average value of 7.64 ___ 0.14 kcal mol-1 for AGx H2° which measures the strength of heme and the folded polypeptide chain. Reaction X ~ D give an average value of 1.56 + 0.08 kcal mo1-1. Since proteins tend to be relatively stable [1], the AG value of 1.56___0.08 kcal mo1-1 is not sufficient to keep the protein in its native form. This seems to suggest that the entire stability of the holoprotein comes from the heme and globin interaction. Both the results of this study and those reported earlier [13,33,34] suggest that the removal of heme should therefore lead to the unfolding of protein, i.e., apocyt c can not exist in the folded conformation.

3.2. Mixed denaturation system

Fig. 4 shows the GdnHCI induced transition curves in presence of various concentrations of CaC12. The pretran-

30

24

b 6 ×

0

~0

0.8

0.6

o

0.4

0.2

0.0

7o7 o :: J / /

0 1 2 3

C

\\ \\\\\\ o \ \ \ \ \

[Gdn HCI ] , M

3

2

O

3

2

r

O ~:

J <~

Fig. 4. GdnHC1 denaturation of cyt c at pH 7.0 and 25 + 0.05°C in presence of 0.0 (-), 0.50 (Q), 1.00 (El), 1.40 (zx) and 2.25 ( 0 ) M CaCI 2. (A) transition curves, (B) normalized transition curves, (C) variation in C,~ values and (D) variation in AG~pp values.


sition region solvent effect are defined by the relation YN = (1860 + 150) X [GdnHCI] + (1570 + 90) X [CaCI2]. It is evident from this relationship that YN depends on both GdnHC1 and CaC12 concentration. A linear extrapolation of results of the posttransition region to 0 M [GdnHCI] gave a value of 23950 + 40 M-x cm-1 for the denatured state in buffer. This value is in excellent agreement with the value obtained by the single system denaturation and with those reported earlier for a random coil cyt c [13,27,28]. This seems to support the idea that the number of denatured states that a protein can give depends upon the folding pattern of the native protein. The reason for saying this is that since cyt c contains only one domain as revealed by X-ray diffraction studies [14,15] only one end product is obtained by different denaturants. Normalized transition curves obtained after defining the solvent effect according to Eq. (3) are shown in Fig. 4B.

Fig. 4C shows the plot of concentration of midpoint, C~ as a function of [CaC12]. The superscript ' c ' represents the presence of CaC12. It can be seen in Fig. 4C that there occurs a sharp decrease in the C c value with an increase in the [CaC12]. Earlier studies with mixed denaturtion suggest that the effect of different denaturants are additive [35,36]. It means due to the additive property the presence of CaC12 enhances the unfolding process so that the denatured state, D is achieved at a lower [GdnHCI].

The Gibbs free energy change (AGapp) plots as a function of [GdnHCl] are shown in Fig. 4D. GdnHC1 induced thermodynamic parameters on the basis of Fig. 4D are as follows: AGanp~ °~ = 6.36 _ 0.13, 5.57 +_ 0.15, 3.23 + 0.25 and 0.68 + 0.23 kcal mol-1; mg = 3.10 _ 0.06, 3.40 _+ 0.09, 2.62 _ 0.21 and 4.11 + 0.88 kca l /mol mol -~', C c = 2.05, 1.64, 1.23 and 0.17 M in presence of 0.50, 1.00, 1.40 and 2.25 M CaC12, respectively.

3.3. Analysis of the results of mixed system

Thermodynamic parameters presented above are due to the combined effect of GdnHC1 and CaC1 e. It can be seen that AG nz°~ values decrease with an increase in [CaCI2]. ----app

Correction of these values for the effect of CaC12 is presented below.

Assuming that both the denaturants of the mixed system cooperate in unfolding of cyt c, we may modify Eq. (10) a s

ZIGap p = AGar@ ° - m g [ g ] - m ~ [ c ] ( 1 1 )

where rag[g] and me[c] represent the GdnHC1 and CaC12 contribution to the m e a s u r e d Z~Gapp, and AGH~ ° is the value o f AGap p in absence of both denaturants at constant temperature an pH. From Eq. (11) it follows that GdnHC1 denaturation can be corrected for the effect of CaC12 in the system. AG~pp, the corrected value of the free energy change can be obtained by the relationship

AGapp = AGapp + mc[C] = ----appAGH20 -- rag[g] (12)

6 =._

O

E 4

2 <I

-2

i

8 •

\ u \ o

No

o

, i r , i

A B

[GdnHCl] .M

' 7

E

og o <~

Fig. 5. Dependence of denaturational free energy, corrected for the effect of CaCI 2 on GdnHCI denaturation. (A) It was assumed that exists no binding site (s) on the protein for the denaturants (B) In the correction it was assumed that the native cyt c contains one binding site for Ca 2-.

we have treated the mixed denaturant results according to Eq. (12) (see Fig. 5A). A least squares analysis of all the data gave a value of 8.69 ___ 0.12 kcal mo1-1 for AGH~ ° and 3.23 + 0.08 kca l /mol mol-1 for m.

In writing Eq. (11) it was assumed that m~ is independent of [CaCI 2 ] and m c is independent of thetGdnHC1]. It is therefore expected that a value of AG H2° = 6.90 + 0.12 - - - app

kcal mo1-1 with mg = 2.70 ___ 0.05 kca l /mol mo1-1 should be obtained after correction, a value obtained from the analysis of the results of the control experiment, i.e., GdnHC1 denaturation in absence of CaCI 2 [13] (also see Fig. 5A, straight line).

From the comparison between expected and observed values it appears that Eq. (11) needs some modification. The reason for saying this is that there may exist binding site(s) for Ca 2÷ on cyt c which imparts stability to native molecule leading to a higher value of AGH~ °. There are two important points to be remembered here (i) It can be seen in Fig. 5 that there appears no nonlinearity in the AG~pp versus [GdnHC1] plot which suggests that GdnHC1 molecule does not have any specific binding site on cyt c molecule. (ii) From the GdnHCI induced transition in presence of different concentrations of KC1 and NaC1 (results not shown here) it is evident that neither the presence of these salts nor their ions (K ÷, Na ÷, C1-) affect the stability of cyt c. This suggests that they do not have any binding site(s) on the protein molecule.

If we take into account that there exists one binding site for Ca 2+, Eq. (11) can be modified as

Aaao p = AGH~ O -- mg[g] - me[el + RT In (1 + ks[C])

(13)

The additional contribution of CaC12 to Z~Gap p values has

230 z. Ahmad, F. Ahmad / Biochimica et Biophysica Acta 1207 (1994) 223-230

been taken into account in Eq. (13). Now we can rearrange the Eq. (13) as

AG~pp / = AGap p d- mc[C ] + R T In (1 + ks[c])

= AGa~ ° - mg[g] (14)

where the superscript ' c o r / ' represents that the b ind ing of Ca 2+ has been introduced.

It can be seen in Fig. 5B that after analyzing the results according to Eq. (14) all points get clustered around the

theoretical l ine drawn with control parameters. A value of 5 tool -1 as the b ind ing constant, k s has been used in adjusting the AGap p values for the presence of CaC12.

Acknowledgments

F.A. is thankful to CSIR (India) for f inancial support

and Z.A. is thankful to professor M. A m i n Head Centre for Biosciences for the Departmental facilities.

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Physico-chemical characterization of products of unfolding of cytochrome c by calcium chloride