Int. J. Peptide Protein Res. 26,1985, 252-261

Equilibrium and kinetic study of sodium- and po tassium-induced conformational changes of apo-a-lactalbumin

Y. HIRAOKA and S . SUGAI

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

Received 19 November 1984, accepted for publication 14 February 1985

Equilibrium and kinetics of Na’and K?-induced conformational changes of apo- a-lactalbumin were studied by means of circular dichroism. While apo-a-lactal- bumin was considerably unfolded in the absence of Na+ or K? in 20 mM Tris at pH 8.0 and 2S0, both the monovalent cations restored the tertiary structure of the protein. Apparent binding constants of Na+ and K‘ to the apoprotein were estimated from the equilibria of the Na+- and K?-induced conformational changes. Based on kinetic data of the conformational changes induced by the monovalent cations, binding mechanism of the ions to the apo-protein was examined. Bound alkali-metal ions stabilize the native-like state and an activated state in the unfolding-refolding reaction of the apoprotein. Key words: bovine apoa-lactalbumin; CD; K+-binding; Na+-binding

a-Lactalbumin is a “modifier” protein in mammary glands, which promotes the synthesis of milk lactose through modification of the substrate specificity of galactosyl transferase (1). A detailed mechanism associated with lactose synthetase activity, however, still remains to be clarified. a-Lactalbumin is evolutionally homologous to lysozyme and their amino acid sequences are strikingly similar (1). But their physicochemical properties are quite different. The equilibrium unfolding of lysozyme by guanidine hydrochloride (GuHCl) is expressed as a two-state transition (2,3). a-

Abbreviations used: CD, circular dichroism; EDTA, ethylenediamine-NNN’N-tetra-acetic acid; EGTA, 2,2’ ethylenedioxybis[ ethyliminodi(acetic acid)] ; GuHC1, guanidine hydrochloride; HEPES, N-2-hydroxyethyl- piperazine-”-2-ethane sulfonic acid.

252

Lactalbumin changes the conformation among three states, involving a stable intermediate, when treated with GuHCl(4). The intermediate state in which the tertiary structure is com- pletely altered but the secondary structure almost intact is termed the “A state”. The A state has a compact structure with a radius of gyration as small as the native state (- 16 A) (5,6). Dolgikh e t al. have suggested that the tertiary structure of the A state fluctuates slowly with a rate constant less than lo-’ s- l and the fluctuation causes the dis- appearance of the aromatic circular dichroism (CD) band (6).

Recently, it has been shown that a- lactalbumin is a Ca2+-binding protein having extremely high affinity for Ca2+ (the binding constant is 106-109 M-’) (7). Removal of the bound CaZ+ markedly destabilizes the native structure of a-lactalbumin (7-9). At

Conformational changes of apo-cu-lactalbumin

In the present work, the equilibrium and kinetics of the conformational changes of bovine apo-cu-lactalbumin induced by Na' and K' were studied by means of CD. The binding parameters of both the ions to the protein are obtained and the binding mechanism is discussed. Also the binding mechanism of Ca2+ is preliminarily compared with that of the monovalent ions.

the present time, it is not clear whether the biological activity of a-lactalbumin is related to the binding of Ca2+ or any other metal ions to the protein. The binding properties of various divalent and trivalent metal ions in a-lactalbumin have been studied by several groups (10-16). There are, however, few studies on the interactions between a- lactalbumin and monovalent metal ions such as Na+ or K'. These two alkali-metal ions are of great significance in biological systems. Further- more, in most of the experiments, the alkali salts are used to keep ionic strength of protein solution. It is, therefore, important to clarify the interactions between the apo-protein and the monovalent cations. In previous work (8), we studied the thermal conformational transition of apo-cu-lactalbumin in a variety of NaCl concentrations and indicated that the thermal stability of the apo-protein strongly depends on the salt concentration. It was concluded that the stabilization is brought about through the binding of Na+ to the vacant Caz+-binding site of the protein. The binding constant of Na' was estimated to bc more than lo2-lo3 M - ' .

0

c - -5

P E u - -10

E" N

(II

U

T 9

X

n (D -15 Y

MATERIALS AND METHODS

Bovine a-lactalbumin was prepared from fresh milk as described previously (4). The Ca2+-free apo-a-lactalbumin was obtained by the method of Hiraoka & Sugai (8). The Ca2+ content in the apo-protein was less than 0.02 per molecule. Concentration of a-lactalbumin was determined spectrophotometrically using a molecular extinction coefficient at 280 nm of 28 500 ~ - l c r n - l (17). All buffer solutions used here were demetalized through a Chelex-1 00 column before use. Contamination of solutions with Ca2+ was checked by a Hitachi 170-10 atomic absorption spectrometer. CD measurements were carried out in a Union CD-1000 or in a

I I I I I I I I I I 21 0 230 250 270 290 310

WAVELENGTH ( nm 1 FIGURE 1 CD spectra of apo-a-lactalbumin at pH 8.0: curve 1, in 2 0 mM Tris in the prescncc of 200 mM NaCl at 25'; 2 , in 20mM Tris in the presence of 1.29mM CaCl, at 25" (native holo-state); 3 , in 20mM Tris in the presence of 500mM KCI at 25"; 4, in 0.2M Tris at 25'; 5 , in 20mM Tris at 25"; 6, in 20mM Trisat 51". Protein concen- tration was 2.3-3.5 X M.

253

Y . Hiraoka and S . Sugai

Jasco J-500A spectropolarimeter, which were calibrated with androsterone (18). In the previous report (8), the calibration was done with d-10-camphor sulfonic acid. The previous CD values for apo-cu-lactalbumin are about 10% less than that in the present study. The path- length of optical quartz cells was 10 mm for the measurements in the region of wavelength 250-350nm and was 1 mrn in 200-250nm. In the equilibrium and kinetic measurements in the presence of NaCl or KCl, salt was added in large excess of the protein concentration. Refolding and unfolding kinetics were deter- mined using stepped increases in the alkali- salt concentration. The method of mixing for the kinetic measurements was described in our previous report (18). The dead time of the mixing was estimated to be less than 5 s. The time constant of the CD apparatuses was set at 1 or 2 s. Parameter optimization was carried out in a microcomputer, CASIO FP-1100.

RESULTS

CD spectra of apo-a-lactalbumin in the absence of metal ions As a buffer species, Tris-HC1 was chosen here, because we can realize the alkali-metal-free state of apo-cu-lactalbumin in the buffer. The

log I - 2.5 -2.0 -1.5 - 1 0

I I I 1

ool -2.5 -2.0 -1 5 -10

l o g [ T r i s l

FIGURE 2 Dependence of CD ellipticity at 270nm of apoa- lactalbumin on Tris concentration at pH 8.0 and 25". Protein concentration was about 3.5 X M. Logarithmic ionic strength (log I) is scaled with pKa value of Tris of 8.06. The signal-to-noise ratios were sufficiently high. The length of each error bar was shorter than the size of each symbol indicating an experimental point.

effect of Tris on the CD spectrum of apo-cu- lactalbumin was first investigated at pH 8.0. The spectra and the dependence of CD ellipticity at 270 nm on Tris concentration are shown in Figs. 1 and 2, respectively. The spectra of apo-a-lactalburnin in 20mM Tris are similar to those of the A state of a-lactalbumin An increase of Tris concentration shifts the CD spectrum of this state towards the spectrum of the native a-lactalbumin. Fig. 3 shows the temperature dependence of the CD ellipticity at 270nm of apo-cu-lactalbumin under various conditions. The metal-free protein exhibits a thermal conformational transition between a native-like state (N) and the A-like state (U) in 0.2 M Tris. These results indicate that Tris affects the stability of the N state of metal- free a-lactalbumin. Therefore, the equilibrium and kinetic experiments of metal binding were carried out in a constant concentration of Tris (20 mM) and at pH 8.0.

- 1 I

I I I I I I 10 20 30 a 0 50 60

TEMPERATURE ( 'C )

FIGURE 3 Thermal change of CD ellipticity at 270 nm of apoa - lactalbumin at pH 8.0: (0 ) . in 20mM Tris in the presence of 200mM NaCl; (o), in 20mM Tris in the presence of 500mM KCI; (O), in 0.2M Tris; (a), in 20 mM Tris. Protein concentrations were ( 0 )

3.38 x 10-5 M, (0) 3.25 x 10-5 M, (0) 3.67 x 10-5 M and (A) 2.27 X lo-' M. Straight lines 1, 2, 3 and 4 are hypothetically drawn for the Na+-complexed, K+complexed and metal-free native-like states, and the metal-free unfolded state of the protein, respect- ivelG Lines 3 and 4 correspond to [ O ] 170 and [ O ] 270 in the text, respectively. The observed value of [0] 2,0 in 20mM Tris at 25" is expected to be equiv- alent to and the hypothetical values of [elflo at 25" in the presence z f the salts are also expected to be equivalent to [0] 270 when K h 4 1 (see text). The levels of experimental error are described in Fig. 2.

N

254

Conformational changes of apo-cu-lactalbumin

where P , M and PM are metal-free protein, metal cation and metal-protein complex, respectively. K:bd is an apparent binding constant.

If the degree of restoration of the tertiary structure, f, is assumed to be proportional to the concentration of the metal-protein complex, then the following relation is obtained:

- - - E o 3

5 -?

b

N

u - N 2

2 X

E a N

- 1 Y

I - 4 - 3 - 2 - 1

Log[Na+l OR Log[K+I

1 Here, [MIf is free metal ion concentration. In terms of CD ellipticity values at 270nm, f is expressed as

[e 1 - [e 1 &o [el go - 161 &o

FIGURE 4 f = The equilibria of conformational changes of apo-or- -

where, [el,",, and [O]zo refer to the extreme lactalbumin in 20mM Tris at pH 8.0 and 25": (o) , induced by Na+; (a), induced by K+. Protein con- centration was about 3.5 X M. Straight lines 1 when [M]f +' and [ M 1 f + m , and 2 refer to the optima values of [el;,, and respectively- [el% denotes the ellipticity [ O ] & for the Na+-induced conformational change, respectively, and the lines 3 and 4 refer to those for K+-induced conformational change. Solid curves are best-fit curves with the optima values of KaPp , [ ~ l ~ , o and [el:,,, listed in Table 1 . The levels of experimental error are described in Fig. 2.

value observed. From eqns- 2 and 3 ,

(4) [el 2070 + K&:d [MI f [dl Go

1 + K$:d [MI

To precisely determine the parameters asso- ciated with the Na' binding, parameter opti-

bind [el i;: =

Binding equilibria of Na' and K' to apo-cu- lactalbumin Sodium- and potassium-induced con formational changes of apo-cu-lactalbumin were observed at 25'. The results are shown in Fig. 4, where the ellipticity at 270nm is plotted as a function of logarithmic concentrations of the alkali-metal ions. The negative ellipticity value increases with an increase of the metal ion concentration and approximates the value for native a- lactalbumin. At high concentrations of the salts, the shapes of the CD spectra also resemble that of the native holo-protein, as seen in Fig. 1. Thus, both Na' and K' appear to restore the native tertiary structure in the apo-protein.

Assuming that the binding of one metal ion per protein molecule is responsible for the conformational change, the binding reaction is expressed by

mization of eqn. 4 was carried out with the non-linear least-squares method. The unknown parameters were K:,$d, [el &O and [e J go. The calculated values of the parameters for the Na' binding are KtGd = 121 f 1 M-' , [el&, = - 7 4 f 1 and [e];, =;350f 1 degcm' dmol-' . The values of [el 279 and [el go are estimated also from the thermal changes of CD ellipticity at 270nm (Fig. 3) and are - 77 and - 347 deg cmz dmol-' , respectively. Thus, the optimum extreme value at [M]f +O agrees well with the value expected from the thermal change of CD ellipticity at 270nm in the absence of the alkali-metal ions, also the extreme value at [MI f + agrees with the value expected from the thermal change in 0.2 M NaCl, and the theoretical curve with the above optima values fits well with the observed points (Fig. 4).

Fig. 4 also indicates that aDo-a-lactalbumin K g d

P + M - PM

I

binds one molecule of K' and acquires the native-like structure as well as in the case of

(1) Na'. The optima values are KtEd = 20.0

255

Y. Hiraoka and S. Sugai

f 0.5 M - ’ , [el &o = - 77 k 1 and [el go = - 345 k 2 deg cmz dmol-’ .

Kinetics of Na+- and K+-induced conformational changes of apo-dactalbumin The kinetics of the alkali-metal-induced con- formational change of apo-cu-lactalbumin were investigated in terms of CD change at 270nm. Rapid increases in the alkali-metal concentration caused unfolding (the negative ellipticity decreased when the alkali-metal concentration was decreased) or refolding (the negative ellipticity increased when the alkali-metal

I I

FIGURE 5 Kinetic progress curves of refolding of apoa-lactal- bumin. The refolding was initiated by a concentration jump of each of the metal ions. A smaU amount of protein solution was injected into an optical cell containing a buffer solution. Protein concentration was 3.3-3.5 X lo-’ M. Na+ concentration was rapidly increased from 0 M to 186 mM (solid curve 1); K+ concentration from 0 M to 456 mM (solid curve 2); Ca2+ concentration from OM to 1.29mM (solid curve 3). An arrow drawn just below “INJECTION” indicates the zero time at which the mixing occurred. Smooth thin curves are drawn theoretically. Straight solid lines and broken lines indicate [ O ] ,,, = 0 and the zero-time extrapolated values of [ O ] , , , of the theoretical curve, respectively.

256

concentration was increased) of the protein. Typical refolding progress curves for Na+- and K+-concentration jumps are shown in Fig. 5. All the unfolding and refolding progress curves obtained can be described a single exponential decay function. The kinetic amplitudes are consistent with the differences in ellipticity expected from the equilibrium Na+- or K’- induced conformational transition curve.

Fig. 6 shows the dependence of the apparent rate constant (kapp) on the final metal concen- tration of the protein solution. The observed rate constant does not depend on the initial metal concentration of the protein solution before the jump. The apparent rate constant shows a minimum just around the midpoint of the Na+- or K+-induced conformational tran- sition of apoa-lactalbumin.

Preliminary kinetic data for calcium-induced refolding of apo-cu-lactalbumin are also illus- trated and compared with the alkali-metal- induced refolding curves in Fig. 5. As clearly seen in Fig. 5 , a major change in CD ellipticity at 270nni occurs within the dead time of the experiment and only a minor change occurs in an observable time range. The major con- formational change induced by Caz+ is far faster than those induced by the alkali-metal ions.

DISCUSSION

Evidence of the binding of alkali-metal ions to apo -@-lac talbum in Apo-cu-lactalbumin is almost in the U state in solutions at low ionic strengths and room temperature. But apo-cu-lactalbumin can form the native-like structure (the N state) without Ca2+ and any other divalent (or trivalent) metal ions. The alkali-metal ions enhanced the stability of the native-like structure of apo-cu- lactalbumin. There are two explanations for the stabilization of the N state at high ionic strengths: (i) the N state is stabilized in solutions at high ionic strengths through the screening of repulsive negative charges in the Ca2+site or on the whole surface of the protein; and (ii) the monovalent cations, Na+ and K’, can bind to the Ca2+-binding site when the site is unoccupied with Caz+ in apo-cu-lactalbumin, and the N conformation is stabilized through the binding of the monovalent cations.

Conformational changes of apo-a-lactalbumin

0 -4 - 3

Log [ Natl Log [ K'I FIGURE 6 Dependence of the apparent rate constant of the conformational changes induced by Na+ and K+ on the final alkali-metal concentration at pH 8.0 and 25". (a), Na+-induced unfolding (0 , B) and refolding (0, 0); initial concentrations of Na+ are (0 ) 200mM. (m) 9.5 mM, (0) 0 M and (D) 9.5 mM. (b), K+-induced unfolding (0, B)

and refolding (0, 0); initial concentration of K+ are (0 ) 500mM, (B) 50mM, (0) O M and ( 0 ) 50mM. Solid and dotted lines are theoretical curves obtained from eqn. 8 under the conditions of Kt # 0 and Kt = 0 by using the parameter values listed in Table 1 , respectively.

Kronman & Bratcher (12) and Hanssens et al. (19) have observed that the difference in ionic strength greatly influences the confor- mational state of apoa-lactalbumin at neutral pH, and they have explained the fact in terms of the electrostatic screening effect. We also have found, in a previous study, that apoa - lactalbumin exhibits the thermal conformational change between the N state and the U state and that the stability of the N state increases with an increase of ionic strength (in NaC1) (8). However, the results were consistent with the mechanism in which the N state can bind preferentially one more Na+ per molecule than the U state. It is noteworthy that the confor- mational changes of the apo-protein caused by different salts (NaCI and KCl) occur in con- siderably different concentration ranges (Fig. 4). In addition, the concentration range of Tris, where the conformational change of the apo- protein occurs, is much more different than those observed for these salts. The fact cannot be interpreted by the electrostatic screening of repulsive negative charges. Also, the screening effect should saturate at lower concentration of these ions than used in this study (20).

According to the 23Na- and j9K-n.m.r. data cited in the report of Lindahl & Vogel (21), Na' and K' can bind to the Caz+-binding site of a-lactalbumin. It has been also shown that the H-n.m.r. spectrum of apoa-lactalbumin in 0.1 M NaCl quite resembles that of the native holo-protein except for small differences in some peaks and is very different from that of the protein in the A state (22,23). Conse- quently, apoa-lactalbumin binds the alkali- metal ions and acquires its specific tertiary structure through this binding.

Kinetic mechanism of metal-induced conformational change of a-lactalbumin Segawa & Sugai (24) have proposed a mechanism for the Caz+-binding of a-lactal- bumin. The mechanism is as follows (Scheme 1): where N, NM, U, and UM correspond to con- formational states, metal-free native(-like), metal-loaded native(-like), metal-free unfolded and metal-loaded unfolded, respectively. The rate constants kl and kz, and the equilibrium constant K h are associated with the confor- mational change between NM and UM. The

257

Y . Hiraoka and S. Sugai

K"

k4

k l

K h

SCHEME 1

constants k S , k 4 , and KO are related to the interconversion between N and U. K t and K r are the binding constants of metal ion to the Caz'-binding site in the N form and U form, respectively. The intrinsic constants are described as

K h = [UM]/[NM] = kz /k l (5)

Kbu = [UMl/[Ul [Mlf

When a sodium or potassium ion binds to the Caz'-binding site, it may be expected that the binding scheme for these metal ions is similar to that for Ca" binding. Therefore, the equilibrium and kinetic data were analyzed on the basis of Scheme 1 .

First, the values of the intrinsic binding constant K f for Na' and K' were evaluated from the binding equilibrium data. Under a condition of K h < 1 (expected from the thermal change of CD ellipticity of apo-cu- lactalbumin in the presence of the metal ions), there is a relationship between the apparent binding constant K:j:d and the above intrinsic constants as follows

KO was calculated by use of the CD data associated with the thermal conformational change of apo-cr-lactalbumin in the absence of Na' and R:

The ellipticity values at 270nm of the metal- free unfolded protein were estimated from the thermal change (Fig. 3). The linear extra- polation of the values on the base line in 20 mM Tris gives a hypothetical value ([el go) of - 55 de cmZ dmol-' at 25'. On the other hand, [el& (= -77,deg cmz dmol-') is the value observed in 20mM Tris at 25'. [ ~ 9 ] $ ~ (= - 313 deg cmz dmol-') was obtained from the thermal CD change in 0.2 M Tris (pH 8.0) because of lower stability of the N state of aposl-lactalbumin in 20 n1M Tris (pH 8.0). Therefore, the calculated value of KO is 10.7. The apparent binding constants, for Na' and K', are 121 f 1 and 20.0 f 0.5 M - ' , respect- ively. Therefore, the intrinsic binding constants, K f k , are obtained to be 1420f 10 M - ' for Na' and 234 f 6 M for K'.

To analyze the kinetic data, it is assumed that the binding processes N-NM, and U-UM are far faster than the interconversions Nt+U and NM-UM (25, 26). Our kinetic data are assumed to be associated with a conformational change between the two kinds of conformational states, {U+UM} and {N+NM} (each state within parentheses is not virtually distinguishable in terms of CD), so that the apparent rate constant kapp for the kinetics is expressed as

and

K f k l / k 3 = K f k z / k d = Kb (9)

where K l is a binding constant of metal ion to an activated state of this protein in the unfolding-refolding reaction. The first term of

258

TA

BL

E 1

Pa

ram

eter

s of

the

equi

libri

um a

nd k

inet

ics

of N

a+- a

nd K

+-in

duce

d con

form

atio

nal c

hang

es o

f apo

-a-la

ctal

bum

in

Liga

nd

Equi

libri

um

Na+

K+

K"

K$

~(M

-,)

K~

(M

-I

)

10.7

12

1 f 1

14

20 f

10

10.7

20

.0*

0.5

234 f 6

Liga

nd

Kin

etic

s (m

odel

la)

K~

(M

-I

)

Kf(

h4

-I)

k, (

s-*)

k

, (s-

1

k, (

s- 1

k, (

s-'

) A

ICC

Na

+ 25

60 f 1

150

2.56

f 1

.46

(15.

6 f 3

5.8)

X l

o-'

(3

.93 f 0

.44)

X l

o-,

(1.7

0 f 0

.16)

X l

o-'

(4.3

1 f 0

.76)

X l

o-'

- 38

9 12

1 f

36

1.69

f 2

.23

(4.1

2 f 1

.95)

X

(7.5

5 f 1

.07)

X

(1.9

3 t

0.35

) X

lo-'

(2

.53 f 0

.29)

X l

o-'

- 3

33

K+

Liga

nd

Kin

etic

s (m

odel

2b)

N-

Ki

(M-'

) K

b (M

'1

N

a+

12.8

f 1

.1

3680

f 1

740

K+

0.82

4 f 0

.133

14

7 f 3

2

k, (

s- '1

k

, (s-

' 1

AIC

' (1

.91 t

0.08

) X

lo-'

(2

.16 f

0.08

) X

lo

-'

(4.6

3 * 1

.12)

X lo

-*

(2.3

6 f 0

.13)

X l

o-'

- 38

9 - 3

34

aMod

el 1

; Kf

#

0 in

eqn

. 8.

bMod

el 2

; Kf

= 0

in e

qn. 8

. A

kaik

e's I

nfor

mat

ion

Cri

teri

on

Y. Hiraoka and S. Sugai

eqn. 8 is related to the refolding rate and the second to the unfolding rate.

When kl > k3 and k2 < k 4 , the function gives a parabola-like curve but with a finite value at each extreme condition [MI f + 0 or [MI -+ 00. Parameter optimization of the kinetic data was carried out on the basis of eqns. 8 and 9 with the non-linear least-squares method. The results of the computation are listed in Table 1, and the best-fit curves are illustrated in Fig. 6. The curves agree well with the experimental values for Na' and K'. The intrinsic binding constant K f , for Na', is in agreement with that obtained from the equilibrium data and, for K', is comparable to that obtained from the equilibrium data. K O ' s

from the ratio of the optimized k3 and k4 are, however, lower than that obtained from the thermal CD change, which may be due to the limited range of the metal concentration in the kinetic measurements.

Because the values of K F are small for Na' and K', the binding of the alkali-metal ions to the U state may be a negligible event. Even if K f = 0, eqn. 8, also gives a parabola-like curve when the ions are assumed to bind to at least the activated state. But the rate constant, k,,,, does not have a finite value when [M]f +-. In order to investigate the significance of the binding to the U state, the curve fitting was carried out under a condition of K F = 0. As seen in Fig. 6 and Table 1, there are no marked differences in terms of the best-fit curve and the values of k 3 , k4 and K f between the first and second optimizations in the experimental concentration ranges of Na' and K'. Also, the values of AIC (Akaike's information criterion), which is a goodness-of-fit criterion (27), are almost the same. It is, therefore, not clear whether the alkali-metal ions bind to the U state. However, it can be concluded that the binding of the alkali-metal ions occurs during the transition from the U state to the activated state and that the alkali-metal ions stabilize not only the N state but also the activated state.

The major conformational change induced by CaZ+ is rapid compared with the alkali- metal-induced conformational changes. Although the rapidity of the Ca2+-induced refolding may be due to the higher stability of

the activated state when Caz+ binds to the state, it should be justified by further kinetic studies with rapid techniques such as stopped flow CD (28).

Relation of the alkali-metal-ion binding to an aspect in the binding of EDTA , EGTA and HEPES in a-lactalbumin When we use chelators such as EDTA or EGTA and also most buffers including Good's buffers, pH's are often adjusted with NaOH or KOH. As described above, Na' and K+ bind to apo-cu- lactalbumin and stabilize the native-like con- formation of the protein. Kronnian and co- workers have reported that HEPES, EDTA and EGTA each bind to apo-cu-lactalbumin and shift its tryptophan fluorescence spectrum towards that of the native protein. In their experiment (1 2), HEPES buffer of 20 mM was used at pH6.5-8.4. In another study (29) they titrated apo-cu-lactalbumin with EDTA or EGTA, the concentration of which was raised up to 0.1 M in each case in 2 0 m M Tris buffer (pH 7.5) . Although they did not describe how they adjusted the pH's of the solutions, they could not have accomplished the pH adjust- ments without a sufficient amount of NaOH or KOH to stabilize the apo-protein. In other words, they did not separate the effect of these organic compounds on the stability of the apo-protein from that of the alkai-metal ion. Therefore, further experiments are required to verify their conclusion that HEPES, EDTA and EGTA can bind to a-lactalbumin and stabilize the N state, considering the presence of Na' or K+ in the protein solutions.

ACKNOWLEDGMENTS

The authors are pleased to thank Drs. K . Kuwajima and K. Nitta for their valuable discussion and Mr. M. Ikeguchi for his comments. This work was sup- ported in part by a Grant-in-Aid of Scientific Research from the Ministry of Education, Japan.

1.

2.

3.

REFERENCES

Hill, R.L. & Brew, K . (1975) Advan. Enzymol. Relat. Areas Mol. Biol. 43,411-490 Tanford, C . (1968) Advan. Protein Chem. 23, 121-282 Tanford, C. (1970) Advan. Protein Chrm. 24, 1-95

260

4. Kuwajima, K., Nitta, K., Yoneyama, M. & Sugai,

5 . Izunii, Y., Miyake, Y., Kuwajima, K., Sugai, S., Inoue, K., Iizumi, M. & Katano, S. (1983) Physica B + C (Amsterdam) 12OB, 444-448

6. Dolgikh, D.A., Cilmanshin, R.I., Brazhnikov, E.V., Bychkova, V.E., Semisotnov, G.V., Venyaminov, S.Y. & Ptitsyn, O.B. (1981) FEBS

7. Hiraoka, Y., Segawa, T., Kuwajima, K., Sugai, S. & Murai, N (1980) Biochem. Biophys. Res. Cotnmun. 95, 1098-1104

8. Hiraoka, Y. & Sugai, S. (1984) Int. J. Pepride Protein Res. 23,535-542

9. Kronman, M.J., Sinha, S.K. & Brew, K. (1981) J. Biol. Chem. 256,8582-8586

10. Permyakov, E.A., Yarmolenko, V.V., Kalini- chenko, L.P., Morozova, L.A. & Burstein, E.A. (1981) Biochem. Biophys. Res. Conimun. 100,

11. Permyakov, E.A., Kalinichenko, L.P., Morozova, L.A., Yarmolenko, V.V. & Burstein, E.A. (1981) Biochem. Biophys. Res. Commun. 102,l-7

12. Kronman, M.J. & Bratcher, S.C. (1984) J. Biol. Chem. 259,10887-10895

13. Bratcher, S.C. & Kronman, M.J. (1984) J. Biol. Chem. 259,10875-10886

14. Murakami, K., Andree, P.J. & Berliner, L.J. (1982) Biochemistry 21,5488-5494

15. Berliner, L.J., Ellis, P.D. & Murakami, K. (1983) Biochemistry 22,s 06 1-5063

16. Bryant, D.T.W. & Andrews, P. (1984) Biochem. J. 220,617-620

17. Kronman, M.J. & Andrcotti, R.E. (1964) Bio- chemistrj) 3,1145-1151

S. (1976) J. Mol. Biol, 106, 359-373

Lett. 136,311-315

191 - 197

Conformational changes of apoa-lactalbumin

18. Kuwajima, K., Hkaoka, Y., Ikeguchi, M. & Sugai, S. (1985) Biochemistry 24, 874-881

19. Hanssens, I., Pottel, H., Herreman, W. & Cauwelaert, F.V. (1984) Biochenz. Biophys. Res. Commun. 119,505-515

20. Tanford C. (1961) Physical Chemistry of Macro- molecules, pp. 457-525, John Wiley & Sons, New York

21. Lindahl, L. & Vogel I-1.J. (1984) Anal. Biochem.

22. Nitta, K., Yokohama, H. & Sugai, S. (1983) Rep. Progr. Polymers 26,599-600

23. Kuwajima, K., Harushima, Y. & Sugai, S . (1985) in preparation

24. Segawa, T. & Sugai, S. (1983) J. Biochem. 93, 1321- 1328

25. Grandjean, I. & Laszlo, P. (1977) FEBS Lett.

26. Williams, R.J.P. (1970) Q. Rev. Chem. Soc. 24, 331-365

27. Nakagawa, T. & Oyanagi, Y. (1982) Data Analysis with the Least-squares Method (in Japanese), pp. 150-156, Tokyo University Press, Tokyo

28. Bayley, P.M. (1981) Progr. Biophys. Mol. Biol.

29. Kronman, M.J. & Bratcher, S.C. (1983) J. Biol. Chem. 258,5707-5709

140,394-402

81,376-380

37,149-180

Address:

Dr. Shintaro Sugai Department of Polymer Science Faculty of Science, Hokkaido University Sapporo, Hokkaido 060 Japan

26 1