Int. J. Peptide Protein Res. 23 , 1984,535-542

Thermodynamics of thermal unfolding of bovine apo-a -1actalbumin

Y . HlRAOKA and S . SUGAI

Department of Polymer Science, Faculty of Science, Hokkaido University, Sapporo, Hokkaido, Japan

Received 27 June, accepted for publication 2 1 October 1983

Thermal unfolding of bovine a-lactalbumin in 10 mM borate buffer at pH 8.0 in the presence of 0 . 0 1 - 1 . 0 ~ NaCl was studied in terms of CD ellipticity. The apoprotein changes the conformation from a native-like (N) to an unfolded (U) form, which has an appreciable amount of the secondary structure but no tertiary structure, in the twostate type. Various thermodynamic parameters of the transition were analyzed. The differences in enthalpy and heat capacity between the N and U states are similar to the corresponding differences of the holoprotein obtained with the calorimetric method by Pfeil. It is shown that one Na+ binds with a binding constant larger than lo2-lo3 M-’ to a specific site (probably to the Ca2+-binding site) in the molecule and the bound Na+ stabilizes the N form of the apoprotein.

Key words: bovine apoa-lactalbumin; Na+-binding; thermal unfolding

a-Lactalbumin is evolutionally homologous to lysozyme and its primary structure is similar to that of lysozyme (1). However, the equi- librium unfolding properties are different. Lysozyme undergoes a two-state unfolding transition by guanidine hydrochloride (GuHCl) (2). a-Lactalbumin changes the conformation among three states, involving a partially unfolded state, when treated with the same denaturant (3). We have characterized the partially unfolded state of bovine, human and goat a-lactalbumins by means of CD (3-6) , ultraviolet absorption (7,8), calorimetric (9), potentiometric (lo), pH-jump (7,8) and ‘H- n.m.r. ( 1 1) measurements. The state is stable at acid pH, and is termed the “A state”. In the A state, the secondary structure remains almost intact, but the tertiary structure disappears. Dolgikh et al. (12) have suggested that the

tertiary structure in the A state fluctuates slowly with a rate constant less than lo’s-’ and that the slow fluctuation causes dis- appearance of the equilibrium CD of the aromatic groups. According to their results, the heat-induced unfolding of bovine and human a-lactalbumins does not bring about the completely unfolded state: they named the heat-induced state the “T state”, which is similar to the A state. We also reported that heat does not induce the complete unfolding of bovine a-lactalbumin (1 3). Pfeil (14) used the calorimetric technique to study the thermal unfolding of the bovine protein and demon- strated that the thermal transition occurs between the two states, the native and the unfolded states.

Recently, another essential difference has been found between lysozyme and a-

535

Y. Hiraoka and S. Sugai

lactalbumin: the native a-lactalbumin is a calcium metalloprotein, while lysozyme does not include any metal ion in the native form (15). The Ca’+ stabilizes the native structure of a-lactalbumin extensively, and removal of Ca’+ produces a state similar to the A state at room temperature. Thus, the heat-induced unfolding of a-lactalbumin may involve elimination of Ca” from the molecule. We analyzed the conformational properties of apo-cu-lactalbumin and the change in its con- formation induced by addition of Ca2+ by means of the CD measurements at 270 nm (16). The results have shown that the apoform itself can assume a native-like conformation at low temperature. The binding constants of the Ca’ + to the native-like apoa-lactalbumins were also determined, by taking the heat-induced conformational change of the apoform into consideration: the constant is very large (10’- 109 ~ - 1 ) .

Although there have been several reports on the conformation of the apoform of a-lactal- bumin (12,15-20), its unfolding and folding processes have not yet been studied. Since the apoprotein assumes the native-like structure at low temperature and the A-like structure at room or high temperature, its thermal unfolding is important to clarify the role of the bound Ca” in the native holoprotein. Moreover, when the conformational stability is compared between lysozyme and a-lactalbumin, it may be preferable to use apoa-lactalbumin.

In this report, the thermodynamic charac- terization of the thermal unfolding of bovine apo-a-lactalbumin is described and the results are compared with those of holoa-lactalbumin.

MATERIALS AND METHODS

Bovine holo-cu-lactalbumin was prepared as des- cribed previously (3,4,7-11). Its apoform was obtained as follows: the holoprotein was dissolved in 4 M GuHCl in 20 mM EDTA-NaOH buffer at pH 8.5. The protein solution was applied on a Sephadex G-25 column equi- librated with 5 mM NH4HC03 buffer (at pH 8.5), which had been demetalized by a Chelex-100 column. Before loading the sample, a small amount of 4M G u H a solution con- taining 20 mM EDTA-NaOH was applied. The

536

apoprotein separated from EDTA, EDTA-CaZ +,

and free Ca” was lyophilized. Content of Ca’+ in the apoform was determined by a Hitachi 170-10 atomic absorption spectrometer, and it was less than 0.02 per molecule. All chemicals used were of reagent grade from Nakarai Chem. Co., Kyoto, Japan.

The buffers used were demetalized through a Chelex-100 column before use. All quartz cells were used after washing with 1 N HCl and rinsing with the demetalized buffers.

Thermal denaturation curve of the apo- protein in 10mM borate buffer-NaC1 was obtained at pH 8.0 by measuring CD at 270 nm in a Union CD-I000 spectropolarimeter. The temperature of the solution was measured by a Takara thermistor thermometer. CD ellip- ticities of the apoform in the far ultraviolet region of 200-250nm were measured in a Jasco 5-20 spectropolarimeter.

The thermodynamic parameters of the thermal unfolding were calculated from the denaturation data with a microcomputer, Casio FP-1100.

RESULTS

Heat-induced change in CD spectrum of bovine apo-a-lactalbumin It has been shown that bovine, human and goat apo-cu-lactalbumins exhibit the thermal unfolding of tertiary structure (16). To get further information about the transition of bovine apoa-lactalbumin, the CD spectra were observed at various temperatures. As shown in Fig. 1, in which the spectra in the borate buffer in the presence of 0.01 M NaCl are shown, the spectrum before the transition (at 11.4’) is very similar to that of native bovine holo-cu-lactalbumin in the near and far ultraviolet regions. The tertiary structure disappears after the transition (at 58’), although an appreciable amount of the secondary structure remains. The apo-a-lactalbumin changes the conformation from the native-like (N) to the partially unfolded (U) state.

A melting curve of the apoprotein is shown in terms of variation in CD ellipticity at 270 nm, [O]270, at ionic strength I = 0.01 in Fig. 2. The ellipticity at 270nm changes abruptly in a narrow region of temperature, and the melting

Thermal unfolding of bovine a-lactalbumin

I I - 0

--loo, I

,-. cl .a m 0

t - -m5 0 -

-a0

220 2 50 280 310 Wavelength [ nm I

FIGURE 1 CD spectra of bovine apoa-lactalbumin in lOmM borate buffer-NaC1 at pH = 8.0, I = 0.01, and tem- peratures 11.4" (--) and 58' (----),where the molecule is in the N and U forms, respectively. Con- centrations of the apoprotein were 7.26 . (for the measurements at 200-250nm) and 3.71 . lo - ' M (at 250-300 nm).

curve is sigmoidal. The CD ellipticity at 222 nm, also changes around the melting tem-

perature T, of the tertiary structure, and this change is followed by further gradual change with increasing temperature.

Dependence of stability of the N form on ionic strength The fraction of the apoprotein in the N state, fN, was calculated from the melting curve at 270nm under the assumption of .the two- state transition:

NU N / U

The fraction fN is expressed as

where [8]& and [8]Ym refer to the N and the U states, respectively. The equilibrium constant KNU (= [U]/[N]) is given by

(3)

The normalized transition curves ( f N vs. temperature T) at various I's are shown in Fig. 3a, and the plots of In KNU vs. 1/T in Fig. 3b. T, depends linearly on log I , as shown in Fig. 4, in which T,'s of the heat- induced transition of the holoprotein are also included. The tertiary structure of the apo- protein is more unstable than that of the holoprotein.

Thermodynamic parameters of the transition The logarithmic equilibrium constant of the thermal unfolding can be expressed by a sum of two terms, according to the theory of protein denaturation (21):

In KNu = ln K k U + ln F(1)

20 40 60 80 Temperature ('C 1

FIGURE 2 Thermal unfolding curves of the apoprotein in 10 mM borate buffer-NaC1 at pH = 8 .0 and I = 0.01. The protein concentrations were the same as in Fig. 1 . Forward ( 0 ) and backward (0) transitions.

537

Y . Hiraoka and S. Sugai

1 0

+=0.5

0

a

.............................

...........................

I 10 20 30 LO 50 60 70 80

Temperature ('C I

FIGURE 3 a: The normalized transition curves of the apoprotein in 1OmM borate buffer-NaC1 at pH = 8.0. I = 0.010 (o), 0.039 (o), 0.057 (a), 0.100 (A), 0.280 (n), 0.505 (=) or 1.000 (0). The best-fitted curve to the experi- mental points at each I is shown. b: Plots of In K N U vs. l /T in the same solution as in (a). Each symbol denotes the same I as in (a).

where F(I) is a function of I alone, and K k U is a characteristic constant of the transition independent of I but dependent on T. Thus, from the plots of In KNU vs. 1/T as various I's, a superposed curve for the temperature

I I - 2 1

LO9 I 0

FIGURE 4 Dependence of T, on I. Open circles refer to the apo- protein and filled triangle and circle show the data of the holoprotein obtained by Pfeil(l4) and Kuwajima & Sugai (1 3), respectively.

3

,

1

3

y ' c c 2

- 1

- 2

- 3 3.0

1 I T 10' I K" 1 b

dependence of l n K N U of the apoprotein at a reference ionic strength can be made by shifting each curve along the l n K N U axis. In Fig. 5, such a superposed curve shifted on the curve at I = 0.1 is shown. The shift factor, S(I), is defined as

s(I) = hl KNu(I)-lll KNU(1 =0.1) (5)

= In F(1)-ln F(I = 0.1)

It is assumed ( 2 2 ) that

In K g U = A + B In T + C/T ( 6 )

where A, B and C are temperature-independent constants. The best-fit values of A + In F(I = O.l ) , B, C and S(1) were calculated using the data shown in Fig. 3b by the linear least-squares method. The superposed curve in Fig. 5 was made with such values of S(1). The enthalpy change AH and the heat capacity change AC, of the N =+ U transition are given by

AH = R(BT- C) and AC, = RB (7)

538

1 / T - 10' I K - ' )

FIGURE 5 The superposed curve of temperature dependence of In K N U at I = 0.1. The curve was made by shifting the ~ K N U - 1/T curves at various I's along the In KNU-axis (see the text). Each symbol used denotes the same I as in Fig. 3.

Thermal unfolding of bovine a-lactalbumin

where R is the gas constant. The ACp and AH values of the apoprotein are listed and com- pared with those of the holoprotein (13,14) in Table 1.

Functional form of F ( I ) Determination of the functional form of F(I) will be useful for clarifying the mechanism of ionic strength-dependent stabilization of native- like apo-a-lactalbumin. The F(I) can be esti- mated from the dependence of the shift factor S on I. The plot of Svs. In I shown in Fig. 6 is linear, indicating that F(I) is also linear to In I, at least in the region of I investigated.

DISCUSSION

An aim of this study is to compare the thermo- dynamic parameters of the thermal unfolding between bovine apo- and holo-a-lactalbumins. The comparison is important for evaluation of the role of the bound Ca2 + to stabilize the native holoprotein. However, it is demon- strated here that the stability of the native-like apoprotein depends considerably on I in aqueous NaCl. The N form is only stable at a temperature less than 0" in the salt-free par- ameters of the apoprotein without salts in the solution. We discuss, firstly, the effect of Na' on stabilization of the native-like apoprotein.

TABLE 1 Comparison of the ACp and A H values of thermal unfolding of bovine a-lactalbumin between the

apo- and holo-forms

Form APO Holo

A C P 4.95 i 0.14a 4.0 i 0.8b 6.48 i 0.1Ec 6.24 i 0.17d

A H e 187 i 18a 130 i 17b

(kJM-' K - ' )

(kJ M-')

10 mM borate with NaCl at I = 0.01-1.0 and pH = 8.0. 4 0 m ~ imidazole-HCI buffer at pH = 6.3 (ref. 14).

'Spectroscopic method with a wavelength 284 nm in 10-20 mM phosphate buffer with KCI of 0.1 Mat pH = 6.8 (ref. 13). dSpectroscopic method with a wavelength 292 nm in 10-20 mM phosphate buffer with KCI of 0.1 Mat pH = 6.8 (ref. 13). eThe values at 25".

539

Y. Hiraoka and S. Sugai

-3 0

- 5 - 4 - 3 -2 1 0 L n I

FIGURE 6 Plot of the shift factor S against I. Solid line is best- fitted to the observed values of S with an assumption of linear dependence of S on 1. Other curves are cal- culated with A n N a = 1 and k = lo3 (--.-), 5 . 10' (----), lo'(-.-) and 10M-' (......).

Effect of Na+ on stabilization of the N form Filimonov et al. (23) showed a similar calcium- binding protein: T, rises from 32" up to 55" after adding 2 M NaCl to parvalbumin solution in 10 mM sodium phosphate and 5 mM EDTA at pH 7.5. The Na+-induced conformational change of parvalbumin or calmodulin was similar to the Ca2+-induced change in terms of difference absorption spectrum (24). Haiech et al. (25) found that K + binds to the CaZ+- binding sites in calmodulin and that the binding constant of K' is 102-103 M-', by use of the flow dialysis technique. Laszlo and co-workers (26--29) indicated more direct evidence of binding of Na+ to the Ca2+-binding sites in parvalbumin, calmodulin and troponin-C with a 23Na-n.m.r. method. Their results of Na+ binding could not be explained by electrostatic condensation on the negatively charged sites in the proteins. The binding constants of Na+ to the sites in the proteins or in the protein fragments including the CaZ +-binding sites were 102-103 M - ' in most cases. Therefore, the monovalent cations can bind to the Ca2 +-

binding sites of the calcium-binding proteins instead of Caz+ and change the local or whole structures of the proteins.

Bovine a-lactalbumin is a calcium-binding protein, and the binding constant of Ca2+ is

as high as those of other typical calcium-binding proteins. Although the structure of the calcium- binding site of the a-lactalbumin remains to be determined, some carboxyl groups may act as the coordination residues (16-20). The calcium- binding site may also bind the monovalent cations such as Na', which contribute to stabil- ization of the native-like structure of apoa - lactalbumin. Because the 10 mM borate buffer does not significantly contribute to I at pH 8.0, I = "a+] in this case. Thus, according to the general theory of ligand-induced conformational change of a protein (21), F(1) and S(1) are expressed as

F(I) = F([Na+]) = (1 + k[Na'])-AnNa (8)

where all of the binding sites are have the same binding constant,

+ k"a+l))

(9)

assumed to k, of Na',

and & I N a is the difference in the number of Na+ bound by the native-like and unfolded forms. "a+] is expressed in mol/l. If we can assume that there is no specific binding of Na+ to the unfolded apoa-lactalbumin, AnNa corresponds to the number of the bound Na+ of the native-like apoa-lactalbumin. When k[Na+] S 1 , S(I) is linear to In I. As shown in Fig. 6, this linearity is held in the concen- tration range of Na+ studied, and the slope,

the other hand, the enthalpy change of the thermal unfolding of a protein at T,, AH(T,), is also related to the number of the binding site of Na+, AnNa, as (21)

dS(I)/d h I , iS -1.27, i.e. & I N a = 1.27. On

The results in Figs. 4 and 5 (at I = 0.1) lead to AnNa = 1.26. Therefore, only one specific Na+-binding site exists in the native-like apo-cu- lactalbumin. Although the exact value of k could not be estimated in this study, the simu- lation curves shown in Fig. 6 indicate that k > 102-103 M-'.

540

Thermal unfolding of bovine a-lactalbumin

a-lactalbumin. It should be clarified in future whether the native structure of a-lactalbumin is less compact than that of lysozyme.

Thermodynamic properties of bovine apo-a- lactalbumin As suggested above, the apoprotein contains one Na+ in the N state in this study. Therefore, In KOw in eqn. 6 should be replaced by In (Kow/k), and the parameters obtained are also affected by the Na+-binding. The previous results of the thermal unfolding of holo-a- lactalbumin (1 3,14) included contributions of the Ca2+-binding. However, comparison of the ACp value in this study and the previous studies of the holoprotein (13,14) may be justified, because the ACp of protein denatur- ation arises mainly from exposure of the hydro- phobic residues in the interior of protein molecule to aqueous medium (21). The value of ACp of apo-a-lactalbumin in this study coincides with that of the holoprotein from the scanning calorimetric data by Pfeil (14), as shown in Table 1. The ACp value from the difference absorption data of the holoprotein (13) is larger than the other values. A change in the Ca2+ activity in the holoprotein solution during the thermal unfolding may affect the denaturation curve, which resulted in a con- siderable change in the ACp value in the last case. The agreement of the ACp value between this and Pfeil's studies shows that the amount of hydrophobic contacts in the native-like apo- protein is similar to that in the native holo- protein.

The enthalpy change of the thermal un- folding of the apoprotein at 25" in this study is similar to that of the holoprotein at 25" obtained by Pfeil (14), as shown in Table 1. Both the values include contributions of the cation binding (Na' and Ca2 +, respectively) in the molecule. However, the heat effect is negligibly small in usual chelation of Ca2+ with oxygen donors and also in the Na+-binding.

As pointed out by Privalov (22), many globular proteins have a characteristic unfolding enthalpy of 13calg-' at 110". The holo-a- lactalbumin has been noted to not have such a value of unfolding enthalpy (14) and it was about 8calg-' at 110". The enthalpy of the apoprotein evaluated in this study is about 10 calg-'. The evolutionally homologous protein, lysozyme, exhibits the characteristic value (1 3 cal g- ' ) at 1 10" for the compact form, larger AH (at 25"), and larger ACp than

ACKNOWLEDGEMENTS

The authors are very grateful to Dr. K . Kuwajima for his valuable discussion and to Drs. K. Nitta and M. Yoneyama for their comments. This work was supported in part by a Grant-in-Aid of Scientific Research from the Ministry o f Education, Japan.

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Address:

Dr. Sci. Shintaro Sugai Dept. of Polymer Science Faculty of Science The University of Hokkaido, Sapporo Japan

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