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Quasielastic light scattering from human α-lactalbumin: comparison of molecular dimensions in native and ‘molten globule’ states
Quasielastic light scattering from human -lactalbumin: comparison of molecular dimensions in native and 'molten globule' states K. Gast*, D. Zirwer*, H. Welfle*, V. E. Bychkova~" and O. B. Ptitsyn~ *Zentralinstitut fiir Molekularbiologie der Akademie der Wissenschaften der DDR, 1115 Berlin-Buch, Robert-R6ssle-Strasse 10, GDR ~flnstitute of Protein Research, USSR Academy of Sciences, 142292 Pushchino, Moscow Region, USSR (Received 23 August 1985; revised 5 March 1986) Quasielastic light scattering has been applied to compare the linear dimensions of human ct-lactalbumin molecules in the native, "molten globule' and unfolded states. The translational diffusion coefficients of the protein have been measured at neutral and acid pH as well as in 6 M guanidine hydrochloride. Temperature dependence of diffusion coefficients for Ca 2 +-free protein at neutral and acid pH have also been obtained. After correction for the protein association, it is shown that the effective linear dimensions of protein molecules increase by about 10% in the 'molten globule' state (i.e. at acid pH or high temperature) as compared with the native state. This increase in linear dimensions is much smaller than the increase in the unfolded state (~40%). Keywords: Protein denaturation; 'moltenglobule'state; ~-lactalbumin;quasielasticlight scattering;translational diffusion coefficient;protein association
Introduction It has been shown that some proteins (e.g. bovine I and human 2 ~-lactalbumin) can exist in a denatured state which substantially differs from the completely disordered one. It has a pronounced secondary structure but has no rigid environment of side groups typical of native proteins. Earlier estimations of the dimensions of bovine ~-lactalbumin molecules in this state (at acid pH), by sedimentation a and electrophoresis '~, suggested a large increase in the molecular volume as compared with the native state. Therefore, it has been assumed 5 that this 'intermediate' state is substantially unfolded but has a well pronounced secondary structure. However, later, it was shown 6-9 that intrinsic viscosities of both bovine and human ~-lactalbumin do not differ very much in their acid and native forms, i.e. the protein molecules remain in the acid form in a more or less compact state. It was shown also 6-9 that ~-lactalbumin molecules in the acid form have no cooperative temperature melting, i.e. they are already 'molten' even at room temperature. In fact, properties of ~-lactalbumin molecules in the temperature-denatured form are similar to their properties in the acid form 6-9, suggesting the idea t h a t both these denatured forms belong to the same 'intermediate' state which has later ~° been called the 'molten globule' state. The value of molecular volume in the 'molten globule' state is very important for the understanding of its physical nature. Intrinsic viscosity data 6-9 for both bovine and human ~-lactalbumin are consistent with the idea that the molecular volume of the 'molten globule' state does not exceed that of the native state by more than 0141--8130/86/0402314)6503.00 © 1986 Butterworth & Co. (Publishers) Ltd
about 30 ~ . However, relatively large experimental errors in the measurements of small intrinsic viscosities do not allow us to evaluate the exact value of this volume. The sedimentation data for bovine 3'9 and human 11 ~lactalbumins are difficult to interpret as they are sensitive to weak association of protein molecules which takes place for bovine ~-lactalbumin in the native form 7'9 and for human ~-lactalbumin in the acid form 7. Gyration radii of bovine a-lactalbumin molecules practically coincide in the native and acid forms 6-9'12'13, but due to weak association of the native protein one cannot exclude a small increase in the molecular volume of the acid form. In this paper, we compare the hydrodynamic effective linear dimensions of human ~-lactalbumin (H~tLA) molecules in different forms by quasielastic light scattering 14'15 (QLS), which allows the measurement of the translational diffusion coefficient of macromolecules with a rather high precision. This technique has been applied successfully to the study of the denaturation of hen egg-white lysozyme by guanidine hydrochloride (GuHCI) 16 as well as to its temperature denaturation 17. Like the methods mentioned above, QLS is sensitive to protein association, too. But combining QLS and integrated light scattering data, the influence of association can be taken into account. We have measured the diffusion coefficients of H~LA in the native, acid and temperature-denatured forms as well as in the form unfolded by GuHC1. It has been shown I s that ~-lactalbumins are Ca 2 ÷-binding proteins and that the removal of the Ca2÷-ion strongly destabilizes their native structure against increasing temperature. Because of the lowered 'melting point', we preferred the Ca 2 ÷ -free form to study the temperature denaturation of H~tLA at neutral pH.
Int. J. Biol. Macromol., 1986, Vol 8, August
231
Quasielastic light scattering from human ~-Iactalbumin: K. Gast et al. Table I
Human ~-lactalbumin: buffer conditions
Form
Buffer
Native
20 mM Tris-HCl, pH 20mM Tris-HCl, pH 50mM KCI-HCI, pH 20ram Tris-HC1, pH hydrochloride
Ca 2 +-free
Acid Unfolded
7.5, 1 mM CaCI2 7.5, 10mM EDTA 2.0 7.5, 6 M guanidine
shown. The integrated scattering intensities were determined in parallel with the QLS measurements in the same apparatus. The native and the unfolded forms were investigated at 20°C. The acid and the Ca z +-free forms were studied at temperatures between 4 and 40°C and between 4 and 50°C, respectively. The diffusion coefficients, D, were calculated from the normalized first order correlation function gtl)(r) =
Experimental Sample preparation Human ~-lactalbumin was prepared from human milk according to Ref. 19 with small modifications. The homogeneity of the protein was checked by electrophoresis in polyacrylamide gradient gels with and without sodium dodecyi sulphate. Four different forms of the protein were studied in buffer conditions as indicated in Table 1. For the QLS experiments of the native form, lyophilized protein was dissolved in Ca2+-containing buffer (Table 1) in order to ensure that the protein molecules do not lose Ca ~ + ions. The protein solution was gel filtrated on a Sephadex G-75 column (2.5 × 100cm). Only peak fractions were used for the measurements. The acid and the CaZ+-free forms were obtained from the native form by dialysis against the respective buffers (Table 1). The unfolded form was prepared by dissolving lyophilized protein in 6 r~ guanidine hydrochloride. The protein concentrations were determined spectrophotometrically using A~'~'~, (280nm)=18.2 or from the dry weight in the case of the unfolded form and were in the range from 3 to 13 mg ml-1 in the QLS experiments.
QLS measurements and data evaluation The QLS measurements were done using a laboratorybuilt apparatus consisting of an argon laser ILA-120 (VEB Carl Zeiss Jena, GDR) operating at 2 = 514.5 nm, a correlator which calculates the 4 bit x 4 bit photon count autocorrelation function at 128 delay times r and a microcomputer system which performs the normalization and the accumulation of the autocorrelation functions. Special care was given to the reduction of light scattering from dust and large particles of aggregated material. We used specially manufactured flow cells to protect the solutions filtered through 0.1 #m Nucleopore filters from any contact with air. The part of the cell which was passed through by the laser beam consisted of a glass tube with a diameter of 7.5 mm. Water, contained in a precision glass vat with a diameter of 120mm, was used as an index matching fluid. The cells were centred within the index matching bath with the help of a special cell holder allowing independent displacements in two perpendicular directions within the scattering plane. The angular uncertainty, mainly determined by the adjustment of the cells, was less than 0.5 degrees. Because of the small diameter, the cells were used only at relatively large scattering angles (0 > 30 degrees). Initially, all four forms of the protein were investigated at scattering angles of 60 and 90 degrees. The measurements yielded identical diffusion coefficients of the protein molecules, within experimental error. Therefore, further investigations (temperature and concentration dependences) were done at 90 degrees and only the results for 90 degrees are
232
Int. J. Biol. Macromol., 1986, Vol 8, August
f]
e x p ( - q2Dz)" F(D)dD
(1)
(q: scattering vector, F(D): distribution function of diffusion coefficients), performing the inverse Laplace transform, using the constrained regularization method z° and the corresponding program Contin 21 of S.W. Provencher. F (D) of our solutions consisted mainly of two peaks, a strong one reflecting the diffusion of protein molecules and a much weaker one caused by the diffusion of buffer molecules, especially of GuHC1. In some cases, a third very small 'dust peak' caused by large particles, which could not be removed by our filtering procedure, had to be taken into account. The diffusion coefficients corresponding to these two additional peaks differ remarkably from that of the protein peak (they are at least five times greater or smaller for buffer and dust, respectively). Because of the small amplitudes of these peaks, the correlation functions have been accumulated until a high signal-to-noise ratio was reached. Thus it was possible to separate these additional contributions well and a true value of the diffusion coefficient of the protein could be evaluated. After extrapolation to zero concentration and correcting to standard conditions, one gets Dz0,w o which will be considered as an apparent diffusion coefficient D,pp of the protein molecules. In particular cases (see Table 2) Dapp is indeed the diffusion coefficient of protein monomers. Corrections jbr protein oligomers The apparent molar masses Mapp (measurement by equilibrium sedimentation ~) of the acid form (16 200g m o l - 1) and the Ca 2 + -free form (17 000 g tool- 1) of H~LA at 20°C are higher than the molar mass calculated from the amino acid sequence (14000g mol-1), while the apparent molar mass of the native form (14 500 g m o l - 1) is practically equal to the calculated one. This points to the presence of a certain number of oligomers in the acid and the Ca 2 + -free forms. These oligomers may be formed by reversible association and/or irreversible aggregation. Both phenomena must be taken into consideration. The amount and the stability of the oligomers in the samples may vary in dependence on buffer conditions, protein concentration and temperature. In any case, these oligomers must be small (dimers or trimers), since the 'protein peak' in F (D) never tends to split in several peaks in our calculations. The diffusion coefficients, D, of small protein oligomers (n < 5) are in the range 0.5"DI
Quasielastic light scattering from human ~-lactalbumin: K. Gast et al. Table 2 Diffusion coefficients and linear expansions of human ct-lactalbumin molecules in different conformational states T
Map p
Form
(°C)
(g mol- I)~
Dapp.X 10 7 (cm2 s- 1)
D] × 107 (cm2 s- ~)
R~ (nm)
S (corrected)
Native
20 4 20 50 20 20
14 500 16 400 17 000 19 600 16 200 -
12.1 10.9 10.4 9.2 10.0 8.6
12.1 11.8 11.4 10.9 10.8 8.6
1.77 1.81 1.88 1.96 1.99 2.47
1.02 1.06 1.11 1.12 1.40
C a 2 + -free
Acid Unfolded
° From Ref. 7 and light scattering data
input data for these calculations are Dapp, Mapp/M~ and X, =D,/D 1..D~ppof a mixture of monomers and one type of polymer, consisting of n of these monomers, measured by QLS, is a z-average
13
•
/f,\
C1M1D 1 + C . M . D .
Dapv=C~MI+C~M'=D1
1+ n l ~ l l ) X ~ l+n(C~-~)
(2)
where C~ and C, are the weight concentrations of monomers and polymers, respectively, M~ and M~ = n M~ their molar masses and X, = D,/DI is the relative decrease in the diffusion coefficients of the polymers as compared with the monomers. The ratio C~/C~ can be calculated from the weight-averaged apparent molar mass
C~M~ + C,M, M~pp
• • •
•
12
÷j + /
I
J
)< ~.11 ~,4
(3)
C ~+ C,
M, pp can be determined either by equilibrium sedimentation or by integrated light scattering. According to equation (3) we get Cn/C I = (Mapp/ M ~) - 1
10
(4)
n - (M~pp/M~ ) Using equations (2) and (4), the diffusion coefficient of monomers D~ can be calculated from Oap p and Mapp/M 1 for a given n. We have performed calculations assuming the presence of either dimers or trimers which yielded similar results. Therefore, the actual type of the oligomers is of minor importance for this correction. Accordingly, only the results for the case of dimers are shown below. Because the intrinsic values of X~ for H~LA oligomers are not available, we used the theoretical values for oligomeric structures of spheres 22 (X 2 =0.718 for n=2). For Mapp/M ~ of the C a 2 +-free and the acid forms of H~LA at 20°C, we used the values measured by equilibrium sedimentation. The data, already published in Ref. 7, are given in Table 2. The sedimentation equilibrium measurements have been c,arried out at 36000 rev/min and at 20°C in a M O M 3170 ultracentrifuge (Hungary) equipped with an interferometric optical system. It took more than 20 h to attain the equilibrium state. Solutions of the acid and the Ca 2 + free forms showed a slight heterogeneity, while homogeneity was observed in the case of the native form. Further experimental details are published in Refs 9 and 11. The temperature dependences of Mapp/M 1 for both forms were calculated from the measured Rayleigh ratio Ro of the solution at a scattering angle 0 = 90 °. Neglecting virial effects R0 = K C M~p~
(5)
~
I
4
~
I
~
8
I
12
~
I
I
16
C (mg-ml- 1 )
Figure I Concentration dependence of the measured diffusion coefficients corrected to standard conditions D2o.wof human ctlactalbumin molecules in the native (m), acid ( + ) and Ca 2 ÷-free (e) forms at 20°C where K = 2nZno2 (c3n/8c)2 2ffl* N21 is the optical constant of the investigated system, no=refraction index of the solvent, ~n/dc=specific increment of refraction index, 20 =wavelength in vacuo, NA = Avogadro's number and C=concentration. It follows that at constant concentration C
Mapp/Ml = Ro/K' (5') where K ' = K M 1 C. Estimated variations of K, which depends on temperature 23, are at most a few per cent within the considered temperature interval. The change in t3n/~3~ is mainly due to changes in the partial specific volume of the protein 23'24. Thus, K (and K') can be considered as temperature-independent and K' can be evaluated from Mapp/M1 measured by equilibrium sedimentation at 20°C. In the case of the acid form only, the neglect of virial effects in equation (5) (see Figure 1) may influence slightly the accuracy of the correction.
Int. J. Biol. Macromol., 1986, Vol 8, August
233
Quasielastic light scatterino .from human ~-Iactalbumin: K. Gast et al. correction for association we obtain apparent values of R (Rapv) and S (Sapp= R a p p / R N ) .
1.4 2.4
[]
1.3
[]
Results
2.2 1.2
2.0 ~ 1.1
I-I[~
0
O0
~.0
0
0
0
0 1.8
-. 1.6
17.9-
a I
I
I
I
I
1.6 + +
1.4
-
m ~
1.2 - .+. +
+
+
+
1.0
I
I
10
20
I
I
I
30
40
50
,
b
T(°C)
Figure 2 (a) Temperature dependence of apparent (~) and corrected (©) swelling factors S and/or corresponding Stokes' radii R of the Ca ~+-free form of human ~-lactalbumin. The experimental values were obtained at C=9.4 mg ml-~ and corrected to zero concentration using the corresponding slope in Fioure 1. (b) Temperature dependence of the ratio Mapp/M~ derived from integrated light scattering Corrections for oligomers have not been considered in the case of H~LA in 6 ~ GuHCI, since H~LA does not associate at high GuHC1 concentrations.
Stokes radii and swellin9 factors The diffusion coefficient D1 can be related to the Stokes' radius R~ by the Stokes-Einstein relation R 1=
kT 6~ulDl
(6)
where k is Boltzmann's constant, T is the absolute temperature and q is the solvent viscosity. We denote D 1 and R 1 of the native protein monomer as DN and R~ and consider, furthermore, all measured values olD and R in relation to these quantities. In particular, we can now introduce a 'swelling factor' S = R I = ON RN
(7)
D1
as a measure of the change in the molecular dimensions compared with the native state. Of course, without
234
Int. J. Biol. Macromol., 1986, Vol 8, August
Figure 1 shows the concentration dependences of the diffusion coefficients D for the native, the acid and the Ca 2 + -free forms. The diffusion coefficient for the unfolded form was obtained at a concentration of 18 mg ml- 1. The typical experimental error in D and in the related quantities is + oj A remarkable concentration _2/o. dependence has been obtained only for the acid form, which probably reflects the electrostatic repulsion of positively charged protein molecules at low pH. The values of Oapp and DI (which coincide in the case of the native and the unfolded forms) are shown in Table 2. Our value of DN for the native H~LA (12.1 x 10 -7 cm 2 s- 1) is practically the same as the value 12.0 x 10- 7 cm z s - 1 (Ref. 25) and greater than the value 10.9 x 10 -7 cm z s i (Ref. 26) obtained earlier by less precise methods. The value of 8.6 x 1 0 - 7 c m 2 s - 1 for the unfolded form is much smaller than the value for the native form. In the case of the C a 2 + free and the acid form, the corrections for oligomers (see Experimental) are necessary. The corrected diffusion coefficients D 1 are, remarkably, greater than the apparent diffusion coefficients Dap p (see Table 2). Figure 2a shows the temperature dependence of the swelling factor and of the Stokes' radius for the Ca 2 +-free H~LA. The squares represent apparent swelling factors Sap p and the corresponding apparent Stokes' radii Rap p without correction for oligomers. The strong dependence of Sapp on temperature has its counterpart in the dependence of Mapp/M 1 o n temperature, derived from measurements of the integrated light scattering intensity (Figure 2b). Both effects were found to be fully reversible. These findings indicate a temperature-dependent association of the Ca2+-free H~LA. Therefore, the corrected values of R and S have been calculated using equations (2), (4), (6) and (7) with Mapp/M~ taken from Figure 2b. It follows that at temperatures below 10°C the Ca 2 +-free form of monomeric H~LA has nearly the same linear dimensions as its native form (Table 2). The linear dimensions of the molecule increase by about 10% in the temperature interval from 10 to 40°C. Figure 3a shows the temperature dependence of the swelling factor and of the Stokes' radius for the acid form of H~LA. In this case, the values of Sapp and Rapp decrease with increasing temperature. This decrease, accompanied by a similar decrease of Mapp/M1 (Figure 3b), reflects the decrease of molecular association. The corrected values of the swelling factors and the Stokes' radii happen to be almost independent of the temperature and show that the linear dimensions of the acid form are about 10% larger than those of the native form. Discussion
The main result of this work is that both acid and temperature denaturation of human ~-lactalbumin lead to an increase in the hydrodynamic effective linear dimensions of the molecules of only ~ 10%, which is much smaller than the increase of --- 40% at its unfolding by 6 M GuHCI. It should be noted that the apparent increase in molecular dimensions without correction for protein
Quasielastic lioht scatterin9 from human ~-lactalbumin: K. Gast et al.
1.25
[]
2.2
[]
1.20
~
Erv
1.15
[] 0
1.10
2.0
0
i l. 9 1.05
I
t
i
t
a
+ +
.~ •
1.1
+
1.0
0
I 10
I 20
I 30
I 40
b
T(*C)
Figure 3 (a) Temperature dependence of apparent (~3) and corrected (©) swelling factors S and/or corresponding Stokes' radii R of the acid form of human ~-iactalbumin. The experimental values were obtained at C= 5.6 mg m1-1 and corrected to zero concentration using the corresponding slope in Fioure I. (b) Temperature dependence of the ratio Mapp/M~ derived from integrated light scattering oligomers is as large as ~ 20% for the acid form (Figure 3a) and even ~ 35% for the temperature-denatured form (Fioure 2a). Therefore, it is worthwhile to dwell on this correction, which considerably reduces the apparent increases. The necessity of these corrections became evident because of the increased values of Mapp and the temperature dependence of the scattering intensity. The integrated scattering intensity of the Ca2+-free form increases by ~ 50% while that of the acid form decreases by ~ 20% upon increasing temperature (see Figures 2b, 3b). According to our estimations (see Experimental), these strong dependences and especially their different signs for the acid and for the Ca 2 +-free forms cannot be explained by temperature-dependent changes in ?n/Oc and/or no. Thus, the only real explanation is a temperature-dependent change of the association-dissociation behaviour of the protein molecules which is different in the acid and in the Ca 2 ÷-free forms. The appropriateness of our corrections is corroborated by two results. First, the corrected diffusion coefficient ( l l . S x 10 -7 cm2s -I) of the Ca2+-free form at 4°C is almost the same as the value of the native form (12.1 x 10- 7 cm 2 s - 1). This result will be discussed later in more
detail. Second, the corrected values of the Stokes' radius of the acid form are nearly independent of temperature. This is consistent with earlier results 8'9 on the absence of the cooperative temperature melting of the acid form. Therefore, we conclude that the real linear expansion of H~LA molecules, both in the acid and the temperaturedenatured form, is about 10%. Our data are consistent with the idea that the structures of the acid and the temperature-denatured forms of ~-lactalbumins are similar 6 9. It should be noted that in the case of temperature denaturation of Ca 2 +-free Hc~LA our data have been derived from the measurement of the same solution (just by heating the scattering cell). Therefore, the relative errors in these data are small (see Figure 2a). The removal of Ca 2 + ions from bovine ~-lactalbumin, taken by itself, does not change its structure, but only decreases its thermostability shifting the midpoint of the temperature transition from 63 to 29°C 9,18. Later, such behaviour was found also for human ~-lactalbumin by circular dichroism measurements (S. Yu. Venyaminov and V. E. Bychkova, unpublished results) and microcalorimetric data 2 v. The removal of Ca 2 + ions from H~LA shifts the midpoint of melting from 65 to 30°C. As the halfwidth of the melting interval of the Ca2 +.free form of H~LA is ~ 20°C, it follows that at 20°C this form is just in the interval of its temperature transition. As a result, the behaviour of this form at 20°C depends on subtle changes in the environment which are difficult to control. Therefore, previously (see Refs. 6, 7) it was concluded that the Ca 2 +-free form of H~LA is almost completely molten even at room temperature. However, under well controlled conditions, as used in this work, this form is only partly molten at room temperature. At temperatures below 10°C, the Ca:+-free form has almost the same linear dimensions as the native form. This agrees well with the results of circular dichroism investigations, In this temperature range, the Ca 2 +-free and the native forms are indistinguishable by their circular dichroism spectra both in the peptide and aromatic regions (S. Yu. Venyaminov and V. E. Bychkova, unpublished results). The corrected values of the Stokes' radii of the Ca 2 ÷ -free form have an sshaped temperature dependence which corresponds to the melting curve of this form. Our results should be compared with earlier data on the linear expansion of a similar protein, lysozyme, at its denaturation. The increase in the linear dimensions of 45% at the unfolding in 6 M GuHC116 is almost the same as for HaLA, while an increase of 18% was found at temperature denaturation ~v. The larger linear expansion of temperature-denatured lysozyme can be explained either by partial association at high temperatures (the authors ~7 themselves discuss t he appearance of associates on heating) or by the fact that temperature denaturation of lysozyme very substantially decreases its secondary structure, while for human ~-lactalbumin a much smaller decrease of secondary structure was found (S. Yu. Venyaminov and V. E. Bychkova, unpublished results). In an accompanying paper 28, it is demonstrated that the acid denaturation of HaLA molecules does not change substantially the intramolecular packing in the core of the protein molecule as compared with the native one (the estimated shift of the mean distance between contacting side chains is as small as ~0.02nm). Further investigations are needed to correlate this small change of macromolecular packing with the 10% expansion of the
Int. J. Biol. Macromol., 1986, Vol 8, August
235
Quasielastic light scattering from human ct-lactalbumin." K. Gast et al. hydrodynamic effective molecular dimensions shown in this paper.
10 11
Acknowledgements
12
The authors thank Dr S. W. Provencher from the Max Planck Institut for biophysikalische Chemie, GiSttingen, FRG, for generously providing the program Contin and N. V. Kotova for technical assistance.
13 14 15 16
References 1 2 3 4 5 6 7 8 9
236
17 18
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Yu. 0., Semisotnov, G. V., Tiktopulo, E. V. and Ptitsyn, O. B. Eur. Biophys. J. 1985, 12, 109 Ohgushi, M. and Wada, A. FEBS Lett. 1983, 164, 21 Dolgikh, D. A. Dissertation (1983) Institute of Protein Research. USSR, Academy of Sciences, Pushchino Iizumi, I., Miyaka, Y., Kuwajima, K., Sugai, S., Inoue, K., lizumi. M. and Katano, S. Physica 1983, 120B, 444 Timchenko, A. A., Dolgikh, D. A., Damaschun, H. and Damaschun, G. Studia biophysica 1986, 112, 201 Berne, B. J. and Pecora, R. 'Dynamic Light Scattering', Wiley and Sons, New York, 1976 Chu, B. 'Laser Light Scattering', Academic Press, New York. 1974 Dubin, S. B., Feher, G. and Benedek, G. B. Biochemi.stry 1973, 12. 714 Nicoli, D. F. and Benedek, G. B. Biopolymers 1976, lfi, 2421 Hiraoka, Y,, Segawa, T., Kuwajima, K., Sugai, S. and Murai, N. Biochem. Biophys. Res. Commun. 1980, 95, 1098 Phillips, N. I. and Jenness, R. Biochim. Biophys. Acta 1971, 229. 407 Provencher, S. W. Comp. Phys~ Commun, 1982, 27, 213 Provencher, S. W. Comp. Phys. Commun, 1982, 27, 229 Garcia Bernal, J, M. and Garcia de la Torre, J. Biopolymers 1981. 20, 129 Huglin, M. B. Ed. 'Light Scattering from Polymer Solutions'. Academic Press, London and New York, 1972, p. 198 Durchschlag, H. and Jaenicke, R. Biochem. Biophys. Res. Commun. 1982, 108, 1074 lvatt, R. J. and Rosemeyer, M. A. Eur. J. Biochem. 1976, 64, 233 Barel, A. O., Prieels, J. P., Maes, E., Looze, Y. and Leonis, J. Biochim. Biophys. Acta 1972, 257, 288 Pfeil, W. and Sadovski, M. L. Studia biophysica 1985, 109, 163 Damaschun, G., Gernat, Ch., Damaschun, H,, Bychkova, V. E. and Ptitsyn, O. B. Int. J. Biol. Macromol. 1986, 8, 226
Introduction It has been shown that some proteins (e.g. bovine I and human 2 ~-lactalbumin) can exist in a denatured state which substantially differs from the completely disordered one. It has a pronounced secondary structure but has no rigid environment of side groups typical of native proteins. Earlier estimations of the dimensions of bovine ~-lactalbumin molecules in this state (at acid pH), by sedimentation a and electrophoresis '~, suggested a large increase in the molecular volume as compared with the native state. Therefore, it has been assumed 5 that this 'intermediate' state is substantially unfolded but has a well pronounced secondary structure. However, later, it was shown 6-9 that intrinsic viscosities of both bovine and human ~-lactalbumin do not differ very much in their acid and native forms, i.e. the protein molecules remain in the acid form in a more or less compact state. It was shown also 6-9 that ~-lactalbumin molecules in the acid form have no cooperative temperature melting, i.e. they are already 'molten' even at room temperature. In fact, properties of ~-lactalbumin molecules in the temperature-denatured form are similar to their properties in the acid form 6-9, suggesting the idea t h a t both these denatured forms belong to the same 'intermediate' state which has later ~° been called the 'molten globule' state. The value of molecular volume in the 'molten globule' state is very important for the understanding of its physical nature. Intrinsic viscosity data 6-9 for both bovine and human ~-lactalbumin are consistent with the idea that the molecular volume of the 'molten globule' state does not exceed that of the native state by more than 0141--8130/86/0402314)6503.00 © 1986 Butterworth & Co. (Publishers) Ltd
about 30 ~ . However, relatively large experimental errors in the measurements of small intrinsic viscosities do not allow us to evaluate the exact value of this volume. The sedimentation data for bovine 3'9 and human 11 ~lactalbumins are difficult to interpret as they are sensitive to weak association of protein molecules which takes place for bovine ~-lactalbumin in the native form 7'9 and for human ~-lactalbumin in the acid form 7. Gyration radii of bovine a-lactalbumin molecules practically coincide in the native and acid forms 6-9'12'13, but due to weak association of the native protein one cannot exclude a small increase in the molecular volume of the acid form. In this paper, we compare the hydrodynamic effective linear dimensions of human ~-lactalbumin (H~tLA) molecules in different forms by quasielastic light scattering 14'15 (QLS), which allows the measurement of the translational diffusion coefficient of macromolecules with a rather high precision. This technique has been applied successfully to the study of the denaturation of hen egg-white lysozyme by guanidine hydrochloride (GuHCI) 16 as well as to its temperature denaturation 17. Like the methods mentioned above, QLS is sensitive to protein association, too. But combining QLS and integrated light scattering data, the influence of association can be taken into account. We have measured the diffusion coefficients of H~LA in the native, acid and temperature-denatured forms as well as in the form unfolded by GuHC1. It has been shown I s that ~-lactalbumins are Ca 2 ÷-binding proteins and that the removal of the Ca2÷-ion strongly destabilizes their native structure against increasing temperature. Because of the lowered 'melting point', we preferred the Ca 2 ÷ -free form to study the temperature denaturation of H~tLA at neutral pH.
Int. J. Biol. Macromol., 1986, Vol 8, August
231
Quasielastic light scattering from human ~-Iactalbumin: K. Gast et al. Table I
Human ~-lactalbumin: buffer conditions
Form
Buffer
Native
20 mM Tris-HCl, pH 20mM Tris-HCl, pH 50mM KCI-HCI, pH 20ram Tris-HC1, pH hydrochloride
Ca 2 +-free
Acid Unfolded
7.5, 1 mM CaCI2 7.5, 10mM EDTA 2.0 7.5, 6 M guanidine
shown. The integrated scattering intensities were determined in parallel with the QLS measurements in the same apparatus. The native and the unfolded forms were investigated at 20°C. The acid and the Ca z +-free forms were studied at temperatures between 4 and 40°C and between 4 and 50°C, respectively. The diffusion coefficients, D, were calculated from the normalized first order correlation function gtl)(r) =
Experimental Sample preparation Human ~-lactalbumin was prepared from human milk according to Ref. 19 with small modifications. The homogeneity of the protein was checked by electrophoresis in polyacrylamide gradient gels with and without sodium dodecyi sulphate. Four different forms of the protein were studied in buffer conditions as indicated in Table 1. For the QLS experiments of the native form, lyophilized protein was dissolved in Ca2+-containing buffer (Table 1) in order to ensure that the protein molecules do not lose Ca ~ + ions. The protein solution was gel filtrated on a Sephadex G-75 column (2.5 × 100cm). Only peak fractions were used for the measurements. The acid and the CaZ+-free forms were obtained from the native form by dialysis against the respective buffers (Table 1). The unfolded form was prepared by dissolving lyophilized protein in 6 r~ guanidine hydrochloride. The protein concentrations were determined spectrophotometrically using A~'~'~, (280nm)=18.2 or from the dry weight in the case of the unfolded form and were in the range from 3 to 13 mg ml-1 in the QLS experiments.
QLS measurements and data evaluation The QLS measurements were done using a laboratorybuilt apparatus consisting of an argon laser ILA-120 (VEB Carl Zeiss Jena, GDR) operating at 2 = 514.5 nm, a correlator which calculates the 4 bit x 4 bit photon count autocorrelation function at 128 delay times r and a microcomputer system which performs the normalization and the accumulation of the autocorrelation functions. Special care was given to the reduction of light scattering from dust and large particles of aggregated material. We used specially manufactured flow cells to protect the solutions filtered through 0.1 #m Nucleopore filters from any contact with air. The part of the cell which was passed through by the laser beam consisted of a glass tube with a diameter of 7.5 mm. Water, contained in a precision glass vat with a diameter of 120mm, was used as an index matching fluid. The cells were centred within the index matching bath with the help of a special cell holder allowing independent displacements in two perpendicular directions within the scattering plane. The angular uncertainty, mainly determined by the adjustment of the cells, was less than 0.5 degrees. Because of the small diameter, the cells were used only at relatively large scattering angles (0 > 30 degrees). Initially, all four forms of the protein were investigated at scattering angles of 60 and 90 degrees. The measurements yielded identical diffusion coefficients of the protein molecules, within experimental error. Therefore, further investigations (temperature and concentration dependences) were done at 90 degrees and only the results for 90 degrees are
232
Int. J. Biol. Macromol., 1986, Vol 8, August
f]
e x p ( - q2Dz)" F(D)dD
(1)
(q: scattering vector, F(D): distribution function of diffusion coefficients), performing the inverse Laplace transform, using the constrained regularization method z° and the corresponding program Contin 21 of S.W. Provencher. F (D) of our solutions consisted mainly of two peaks, a strong one reflecting the diffusion of protein molecules and a much weaker one caused by the diffusion of buffer molecules, especially of GuHC1. In some cases, a third very small 'dust peak' caused by large particles, which could not be removed by our filtering procedure, had to be taken into account. The diffusion coefficients corresponding to these two additional peaks differ remarkably from that of the protein peak (they are at least five times greater or smaller for buffer and dust, respectively). Because of the small amplitudes of these peaks, the correlation functions have been accumulated until a high signal-to-noise ratio was reached. Thus it was possible to separate these additional contributions well and a true value of the diffusion coefficient of the protein could be evaluated. After extrapolation to zero concentration and correcting to standard conditions, one gets Dz0,w o which will be considered as an apparent diffusion coefficient D,pp of the protein molecules. In particular cases (see Table 2) Dapp is indeed the diffusion coefficient of protein monomers. Corrections jbr protein oligomers The apparent molar masses Mapp (measurement by equilibrium sedimentation ~) of the acid form (16 200g m o l - 1) and the Ca 2 + -free form (17 000 g tool- 1) of H~LA at 20°C are higher than the molar mass calculated from the amino acid sequence (14000g mol-1), while the apparent molar mass of the native form (14 500 g m o l - 1) is practically equal to the calculated one. This points to the presence of a certain number of oligomers in the acid and the Ca 2 + -free forms. These oligomers may be formed by reversible association and/or irreversible aggregation. Both phenomena must be taken into consideration. The amount and the stability of the oligomers in the samples may vary in dependence on buffer conditions, protein concentration and temperature. In any case, these oligomers must be small (dimers or trimers), since the 'protein peak' in F (D) never tends to split in several peaks in our calculations. The diffusion coefficients, D, of small protein oligomers (n < 5) are in the range 0.5"DI
Quasielastic light scattering from human ~-lactalbumin: K. Gast et al. Table 2 Diffusion coefficients and linear expansions of human ct-lactalbumin molecules in different conformational states T
Map p
Form
(°C)
(g mol- I)~
Dapp.X 10 7 (cm2 s- 1)
D] × 107 (cm2 s- ~)
R~ (nm)
S (corrected)
Native
20 4 20 50 20 20
14 500 16 400 17 000 19 600 16 200 -
12.1 10.9 10.4 9.2 10.0 8.6
12.1 11.8 11.4 10.9 10.8 8.6
1.77 1.81 1.88 1.96 1.99 2.47
1.02 1.06 1.11 1.12 1.40
C a 2 + -free
Acid Unfolded
° From Ref. 7 and light scattering data
input data for these calculations are Dapp, Mapp/M~ and X, =D,/D 1..D~ppof a mixture of monomers and one type of polymer, consisting of n of these monomers, measured by QLS, is a z-average
13
•
/f,\
C1M1D 1 + C . M . D .
Dapv=C~MI+C~M'=D1
1+ n l ~ l l ) X ~ l+n(C~-~)
(2)
where C~ and C, are the weight concentrations of monomers and polymers, respectively, M~ and M~ = n M~ their molar masses and X, = D,/DI is the relative decrease in the diffusion coefficients of the polymers as compared with the monomers. The ratio C~/C~ can be calculated from the weight-averaged apparent molar mass
C~M~ + C,M, M~pp
• • •
•
12
÷j + /
I
J
)< ~.11 ~,4
(3)
C ~+ C,
M, pp can be determined either by equilibrium sedimentation or by integrated light scattering. According to equation (3) we get Cn/C I = (Mapp/ M ~) - 1
10
(4)
n - (M~pp/M~ ) Using equations (2) and (4), the diffusion coefficient of monomers D~ can be calculated from Oap p and Mapp/M 1 for a given n. We have performed calculations assuming the presence of either dimers or trimers which yielded similar results. Therefore, the actual type of the oligomers is of minor importance for this correction. Accordingly, only the results for the case of dimers are shown below. Because the intrinsic values of X~ for H~LA oligomers are not available, we used the theoretical values for oligomeric structures of spheres 22 (X 2 =0.718 for n=2). For Mapp/M ~ of the C a 2 +-free and the acid forms of H~LA at 20°C, we used the values measured by equilibrium sedimentation. The data, already published in Ref. 7, are given in Table 2. The sedimentation equilibrium measurements have been c,arried out at 36000 rev/min and at 20°C in a M O M 3170 ultracentrifuge (Hungary) equipped with an interferometric optical system. It took more than 20 h to attain the equilibrium state. Solutions of the acid and the Ca 2 + free forms showed a slight heterogeneity, while homogeneity was observed in the case of the native form. Further experimental details are published in Refs 9 and 11. The temperature dependences of Mapp/M 1 for both forms were calculated from the measured Rayleigh ratio Ro of the solution at a scattering angle 0 = 90 °. Neglecting virial effects R0 = K C M~p~
(5)
~
I
4
~
I
~
8
I
12
~
I
I
16
C (mg-ml- 1 )
Figure I Concentration dependence of the measured diffusion coefficients corrected to standard conditions D2o.wof human ctlactalbumin molecules in the native (m), acid ( + ) and Ca 2 ÷-free (e) forms at 20°C where K = 2nZno2 (c3n/8c)2 2ffl* N21 is the optical constant of the investigated system, no=refraction index of the solvent, ~n/dc=specific increment of refraction index, 20 =wavelength in vacuo, NA = Avogadro's number and C=concentration. It follows that at constant concentration C
Mapp/Ml = Ro/K' (5') where K ' = K M 1 C. Estimated variations of K, which depends on temperature 23, are at most a few per cent within the considered temperature interval. The change in t3n/~3~ is mainly due to changes in the partial specific volume of the protein 23'24. Thus, K (and K') can be considered as temperature-independent and K' can be evaluated from Mapp/M1 measured by equilibrium sedimentation at 20°C. In the case of the acid form only, the neglect of virial effects in equation (5) (see Figure 1) may influence slightly the accuracy of the correction.
Int. J. Biol. Macromol., 1986, Vol 8, August
233
Quasielastic light scatterino .from human ~-Iactalbumin: K. Gast et al. correction for association we obtain apparent values of R (Rapv) and S (Sapp= R a p p / R N ) .
1.4 2.4
[]
1.3
[]
Results
2.2 1.2
2.0 ~ 1.1
I-I[~
0
O0
~.0
0
0
0
0 1.8
-. 1.6
17.9-
a I
I
I
I
I
1.6 + +
1.4
-
m ~
1.2 - .+. +
+
+
+
1.0
I
I
10
20
I
I
I
30
40
50
,
b
T(°C)
Figure 2 (a) Temperature dependence of apparent (~) and corrected (©) swelling factors S and/or corresponding Stokes' radii R of the Ca ~+-free form of human ~-lactalbumin. The experimental values were obtained at C=9.4 mg ml-~ and corrected to zero concentration using the corresponding slope in Fioure 1. (b) Temperature dependence of the ratio Mapp/M~ derived from integrated light scattering Corrections for oligomers have not been considered in the case of H~LA in 6 ~ GuHCI, since H~LA does not associate at high GuHC1 concentrations.
Stokes radii and swellin9 factors The diffusion coefficient D1 can be related to the Stokes' radius R~ by the Stokes-Einstein relation R 1=
kT 6~ulDl
(6)
where k is Boltzmann's constant, T is the absolute temperature and q is the solvent viscosity. We denote D 1 and R 1 of the native protein monomer as DN and R~ and consider, furthermore, all measured values olD and R in relation to these quantities. In particular, we can now introduce a 'swelling factor' S = R I = ON RN
(7)
D1
as a measure of the change in the molecular dimensions compared with the native state. Of course, without
234
Int. J. Biol. Macromol., 1986, Vol 8, August
Figure 1 shows the concentration dependences of the diffusion coefficients D for the native, the acid and the Ca 2 + -free forms. The diffusion coefficient for the unfolded form was obtained at a concentration of 18 mg ml- 1. The typical experimental error in D and in the related quantities is + oj A remarkable concentration _2/o. dependence has been obtained only for the acid form, which probably reflects the electrostatic repulsion of positively charged protein molecules at low pH. The values of Oapp and DI (which coincide in the case of the native and the unfolded forms) are shown in Table 2. Our value of DN for the native H~LA (12.1 x 10 -7 cm 2 s- 1) is practically the same as the value 12.0 x 10- 7 cm z s - 1 (Ref. 25) and greater than the value 10.9 x 10 -7 cm z s i (Ref. 26) obtained earlier by less precise methods. The value of 8.6 x 1 0 - 7 c m 2 s - 1 for the unfolded form is much smaller than the value for the native form. In the case of the C a 2 + free and the acid form, the corrections for oligomers (see Experimental) are necessary. The corrected diffusion coefficients D 1 are, remarkably, greater than the apparent diffusion coefficients Dap p (see Table 2). Figure 2a shows the temperature dependence of the swelling factor and of the Stokes' radius for the Ca 2 +-free H~LA. The squares represent apparent swelling factors Sap p and the corresponding apparent Stokes' radii Rap p without correction for oligomers. The strong dependence of Sapp on temperature has its counterpart in the dependence of Mapp/M 1 o n temperature, derived from measurements of the integrated light scattering intensity (Figure 2b). Both effects were found to be fully reversible. These findings indicate a temperature-dependent association of the Ca2+-free H~LA. Therefore, the corrected values of R and S have been calculated using equations (2), (4), (6) and (7) with Mapp/M~ taken from Figure 2b. It follows that at temperatures below 10°C the Ca 2 +-free form of monomeric H~LA has nearly the same linear dimensions as its native form (Table 2). The linear dimensions of the molecule increase by about 10% in the temperature interval from 10 to 40°C. Figure 3a shows the temperature dependence of the swelling factor and of the Stokes' radius for the acid form of H~LA. In this case, the values of Sapp and Rapp decrease with increasing temperature. This decrease, accompanied by a similar decrease of Mapp/M1 (Figure 3b), reflects the decrease of molecular association. The corrected values of the swelling factors and the Stokes' radii happen to be almost independent of the temperature and show that the linear dimensions of the acid form are about 10% larger than those of the native form. Discussion
The main result of this work is that both acid and temperature denaturation of human ~-lactalbumin lead to an increase in the hydrodynamic effective linear dimensions of the molecules of only ~ 10%, which is much smaller than the increase of --- 40% at its unfolding by 6 M GuHCI. It should be noted that the apparent increase in molecular dimensions without correction for protein
Quasielastic lioht scatterin9 from human ~-lactalbumin: K. Gast et al.
1.25
[]
2.2
[]
1.20
~
Erv
1.15
[] 0
1.10
2.0
0
i l. 9 1.05
I
t
i
t
a
+ +
.~ •
1.1
+
1.0
0
I 10
I 20
I 30
I 40
b
T(*C)
Figure 3 (a) Temperature dependence of apparent (~3) and corrected (©) swelling factors S and/or corresponding Stokes' radii R of the acid form of human ~-iactalbumin. The experimental values were obtained at C= 5.6 mg m1-1 and corrected to zero concentration using the corresponding slope in Fioure I. (b) Temperature dependence of the ratio Mapp/M~ derived from integrated light scattering oligomers is as large as ~ 20% for the acid form (Figure 3a) and even ~ 35% for the temperature-denatured form (Fioure 2a). Therefore, it is worthwhile to dwell on this correction, which considerably reduces the apparent increases. The necessity of these corrections became evident because of the increased values of Mapp and the temperature dependence of the scattering intensity. The integrated scattering intensity of the Ca2+-free form increases by ~ 50% while that of the acid form decreases by ~ 20% upon increasing temperature (see Figures 2b, 3b). According to our estimations (see Experimental), these strong dependences and especially their different signs for the acid and for the Ca 2 +-free forms cannot be explained by temperature-dependent changes in ?n/Oc and/or no. Thus, the only real explanation is a temperature-dependent change of the association-dissociation behaviour of the protein molecules which is different in the acid and in the Ca 2 ÷-free forms. The appropriateness of our corrections is corroborated by two results. First, the corrected diffusion coefficient ( l l . S x 10 -7 cm2s -I) of the Ca2+-free form at 4°C is almost the same as the value of the native form (12.1 x 10- 7 cm 2 s - 1). This result will be discussed later in more
detail. Second, the corrected values of the Stokes' radius of the acid form are nearly independent of temperature. This is consistent with earlier results 8'9 on the absence of the cooperative temperature melting of the acid form. Therefore, we conclude that the real linear expansion of H~LA molecules, both in the acid and the temperaturedenatured form, is about 10%. Our data are consistent with the idea that the structures of the acid and the temperature-denatured forms of ~-lactalbumins are similar 6 9. It should be noted that in the case of temperature denaturation of Ca 2 +-free Hc~LA our data have been derived from the measurement of the same solution (just by heating the scattering cell). Therefore, the relative errors in these data are small (see Figure 2a). The removal of Ca 2 + ions from bovine ~-lactalbumin, taken by itself, does not change its structure, but only decreases its thermostability shifting the midpoint of the temperature transition from 63 to 29°C 9,18. Later, such behaviour was found also for human ~-lactalbumin by circular dichroism measurements (S. Yu. Venyaminov and V. E. Bychkova, unpublished results) and microcalorimetric data 2 v. The removal of Ca 2 + ions from H~LA shifts the midpoint of melting from 65 to 30°C. As the halfwidth of the melting interval of the Ca2 +.free form of H~LA is ~ 20°C, it follows that at 20°C this form is just in the interval of its temperature transition. As a result, the behaviour of this form at 20°C depends on subtle changes in the environment which are difficult to control. Therefore, previously (see Refs. 6, 7) it was concluded that the Ca 2 +-free form of H~LA is almost completely molten even at room temperature. However, under well controlled conditions, as used in this work, this form is only partly molten at room temperature. At temperatures below 10°C, the Ca:+-free form has almost the same linear dimensions as the native form. This agrees well with the results of circular dichroism investigations, In this temperature range, the Ca 2 +-free and the native forms are indistinguishable by their circular dichroism spectra both in the peptide and aromatic regions (S. Yu. Venyaminov and V. E. Bychkova, unpublished results). The corrected values of the Stokes' radii of the Ca 2 ÷ -free form have an sshaped temperature dependence which corresponds to the melting curve of this form. Our results should be compared with earlier data on the linear expansion of a similar protein, lysozyme, at its denaturation. The increase in the linear dimensions of 45% at the unfolding in 6 M GuHC116 is almost the same as for HaLA, while an increase of 18% was found at temperature denaturation ~v. The larger linear expansion of temperature-denatured lysozyme can be explained either by partial association at high temperatures (the authors ~7 themselves discuss t he appearance of associates on heating) or by the fact that temperature denaturation of lysozyme very substantially decreases its secondary structure, while for human ~-lactalbumin a much smaller decrease of secondary structure was found (S. Yu. Venyaminov and V. E. Bychkova, unpublished results). In an accompanying paper 28, it is demonstrated that the acid denaturation of HaLA molecules does not change substantially the intramolecular packing in the core of the protein molecule as compared with the native one (the estimated shift of the mean distance between contacting side chains is as small as ~0.02nm). Further investigations are needed to correlate this small change of macromolecular packing with the 10% expansion of the
Int. J. Biol. Macromol., 1986, Vol 8, August
235
Quasielastic light scattering from human ct-lactalbumin." K. Gast et al. hydrodynamic effective molecular dimensions shown in this paper.
10 11
Acknowledgements
12
The authors thank Dr S. W. Provencher from the Max Planck Institut for biophysikalische Chemie, GiSttingen, FRG, for generously providing the program Contin and N. V. Kotova for technical assistance.
13 14 15 16
References 1 2 3 4 5 6 7 8 9
236
17 18
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Yu. 0., Semisotnov, G. V., Tiktopulo, E. V. and Ptitsyn, O. B. Eur. Biophys. J. 1985, 12, 109 Ohgushi, M. and Wada, A. FEBS Lett. 1983, 164, 21 Dolgikh, D. A. Dissertation (1983) Institute of Protein Research. USSR, Academy of Sciences, Pushchino Iizumi, I., Miyaka, Y., Kuwajima, K., Sugai, S., Inoue, K., lizumi. M. and Katano, S. Physica 1983, 120B, 444 Timchenko, A. A., Dolgikh, D. A., Damaschun, H. and Damaschun, G. Studia biophysica 1986, 112, 201 Berne, B. J. and Pecora, R. 'Dynamic Light Scattering', Wiley and Sons, New York, 1976 Chu, B. 'Laser Light Scattering', Academic Press, New York. 1974 Dubin, S. B., Feher, G. and Benedek, G. B. Biochemi.stry 1973, 12. 714 Nicoli, D. F. and Benedek, G. B. Biopolymers 1976, lfi, 2421 Hiraoka, Y,, Segawa, T., Kuwajima, K., Sugai, S. and Murai, N. Biochem. Biophys. Res. Commun. 1980, 95, 1098 Phillips, N. I. and Jenness, R. Biochim. Biophys. Acta 1971, 229. 407 Provencher, S. W. Comp. Phys~ Commun, 1982, 27, 213 Provencher, S. W. Comp. Phys. Commun, 1982, 27, 229 Garcia Bernal, J, M. and Garcia de la Torre, J. Biopolymers 1981. 20, 129 Huglin, M. B. Ed. 'Light Scattering from Polymer Solutions'. Academic Press, London and New York, 1972, p. 198 Durchschlag, H. and Jaenicke, R. Biochem. Biophys. Res. Commun. 1982, 108, 1074 lvatt, R. J. and Rosemeyer, M. A. Eur. J. Biochem. 1976, 64, 233 Barel, A. O., Prieels, J. P., Maes, E., Looze, Y. and Leonis, J. Biochim. Biophys. Acta 1972, 257, 288 Pfeil, W. and Sadovski, M. L. Studia biophysica 1985, 109, 163 Damaschun, G., Gernat, Ch., Damaschun, H,, Bychkova, V. E. and Ptitsyn, O. B. Int. J. Biol. Macromol. 1986, 8, 226