- Email: [email protected]
Study of pressure-induced denaturation of bovine serum albumin by photon correlation spectroscopy
Volun~c 9 I _ number 3
CHEMICAL
PHYSICS LETTERS
STUDY OF PRESSURE-INDUCED DENATURATION
10
September
1982
OF BOVlNE SERUM ALBUMIN
BY PHOTON CORRELATION SPECTROSCOPY
B. NYSTROhl l and J ROOTS ** b~srrrrrrcof ~I~~rcalClrmnrsrr~~,Unrwrsrry
of Uppsala. P 0. Bor 532, S-751 -71 Uppsala. Swederr
Rcccircd 10 June 1982, m fin.11form 3 July 1982
The prcssurc-mduced dcnawnrlon al bovine serum albums (BSA) m w-buffer atpH 7.4 was studied m the pressure rang l-4500 alni usrng the photon correlallon specrroscopy technique. The mfluence of pressure on the hydrodynamic radius and on the dtifuslon cocfficwnl was studled
1. introduction The nnllve structure of a protem 1s often stable over a farly wide range of external conditions, but It can be disrupted by sufficiently drastic changes III phys~al or chenucal environment. The process, during which the folded structure of a protem is changed mto the unfolded form, is known as dmmwu~wt~ In the past the denaturation of common protems has been studied using a variety of denaturing agents such as pH, heat, pressure, and chemical denaturants [ 11. There has been a moderate amount of work on the pressure-mduced denaturation of protem molecules in solutlon [2-IO]. Mo$t of these publications have been concerned with the influence of pressure on therm@ dynamic and related quantities, there is a lack of investigations
de&g
with the mfluence of pressure on
transport and dynanuc propertres. In a recent investigation [ 111 we used photon correlauon spectroscopy (PCS) to study the pressureinduced denaturation
of bovme serum albumn
(BSA)
III acetate
buffer with added salt (~OIUC strength I Z= 0. I2 hi) at the lsoelectrlcpH(4.7) and in the pressure interval l-4000 atm. However, there are some factors
* Presentaddress’Depdrtmentof Chemistry,The Uruverslty of Oslo, P.O. Box 1033, Blmdem, Oslo 3. Noruy.
** Presentaddress Chemlal UK.
236
Department of Chemical Engmekng and Technology, ImpcrwlCollege, London SW7 2BY,
which may constitute complications in the analysis of data. Firstly, the employedpH and ionic strength are expected to represent a situation where the electrostatic forces are strongly screened, there are indications III the Merature [ 12,131 that such expenmental conditions favour formation of aggregates in protein solutions. Secondly, it has been shown 1141 that pH of an acetate buffer decreases by approximately one pH unit over a pressure range of 6500 atm. In mew of these aspects, and the knowledge that the features of the denaturation process usually are affected by environmental alteratrons, such aspH and ionic strength, we decided to perform measurements under different and more well-defmed expenmental conditions. The sun of this investigation IS to study how the pressure-induced denaturation affects the size and the diffusion behaviour of the protein molecules. By USIII~ pressure as the denaturant we may learn more about the factors which determme the structure and stablty of protein molecules. In the present work we have studied the pressure denahrrauon of BSA m a MS-buffer (no salt was added) of ionic strength 0.04 M and at pH = 7.4 by means of PCS. At these conditions electrostatic repulsions between the BSA molecules can be expected; possible aggregation effects should be suppressed. In addition, it has been observed [14] that pH of a trisbuffer solution is practically insensitive to pressure in the interval I-6500 atm.
0 009-2614/82/0000-0000/%
02.75 0 1982 North-Holland
Volume 91, number
3
CHEMICAL
PHYSICS LITTERS
IO Scptcmbcr 1987
2. Experimental
gle-mode operation. Light scattered at a scattering
2.1 Matenalr atrd preparatrorl olsolutiorls
angle 0 = 90” was imaged on the photocathode of an In FW130 photomultipher tube. The resulting pulses
A sulfhydryl blocked BSA monomer sample (characterized as 99% monomer) obtained as standard protein powder from hZlles Laboratories (stock no. 81-028-2) was utilized in these experiments. The pH of the protein solutions was adjusted to pH = 7.4 in a t&buffer of ionic strength 0.04 M. BSA concentrations (c) were determined spectrophotometrically using a value for the extmction coefficient, Et?&, of 6.67 mol-’ cm-’ [IS] at 280 MI. The solutions were filtered through 0.22 Mm Mdlipore falters directly mto pre-cleaned hght scattering cuvettes of 16 mm path length in an atmosphere of filtered air. The solutions were kept at 4’C until use and all measurements were taken within four days of preparation of the BSA monomer solutions. 2 2. Eq~t~pmer~tand data analysis The high-pressure vessel has been described in detd elsewhere [ 161. The equipment used in the present PCS measurements was the same as that descriied previously [11,17J. A three-window portable optical high-pressure vessel 1sused, cor.tairtmg the sample solution within a cylindrical quartz cuvette, which has an optically flat bottom and two oppositely placed, optIcally flat wmdows; the cuvette was closed with a movable blackoxidized alummmm plunger, which transmits the pressure and isolates the sample soUlon from the pressure-transmitting fluid glycerol Pressure is applied
with a small hydraulic press, equipped with a Bourdon gauge which has been accurately cahbrated. In ail measurements, the pressure is slowly increased and the sample solution is allowed to equilibrate (2-3 h) before data are accumulated over an interval of l-5 mm. The h&pressure cell is enclosed in an insuiatmg Jacket, through which a water-ethanol mixture is CLTculated from a thermostatted bath. By this arrangement the temperature in the sample cuvette could be maintained constant within +O.l’C. The measurements were carried out at 25’C. A Coherent Radiation model CR4 argon ion laser was used at a wavelength of 488 nm with an etalon frequency stabilizer in the optical cavity to ensure sin-
were amphfied and discriminated and then fed mto a 128.channel dlgitti correlator (Langley-Ford Instruments) wh~h was set to generate tbefiilf autocorrelation function of the scattered mtensity. The correlator was mterfaced to a Luxor ABC 80 microcomputer, which was programmed to calculate the normahzed full-photon counting time correlation function, and to analyse and store data. In tlus study the homodyne configuration was bsed. Intensity correlation data were routinely analysed usmg basically the method of cumulants [18,19] III order to provide the average decay rate r and the normalized variance Q; this latter quantity is a measure of the width of the btribution of the decay rates. It was found that the experimental correlation functions could be well represented by single exponentlals (Q < O.OS),except for those representing the highest concentration at the highest pressures, where increasing departure from “single exponentiahty” was observed. The chffusion coefficient D was determined from the average decay rate r according to i==Dq’,
(1)
where q =(471/A) H sm(O/?,) 1sthe magnitude of the scattering vector, X is the incident light wavelength, II is the index of refraction of solution, and 0 is the scattenng angle. In this study II was determmed from the measured refractive index of solution and the known 1201 pressure dependence of the index of refractlon of WPflx.
3. Results and discussion
Photon cocrelatton spectroscopy constitutes a powerful tool to study diffusion as well as to determine the actual average size change of the protem molecules during the denaturation process. The effective hydrodynamic radius RH may be obtamed from the StokesEmstein relation RH = kT/6nqD ,
(2)
where k is Boltzmann’s constant, T is the absolute temperature, and q is the solvent viscosity. In this work, values of q at tiferent pressures were estunated 237
\‘olume 9 1. nulllber 3
CliCXIiCAl.
PHYSICS
from the vtscosity of the buffer system and the reported [Z I j pressure dependence of the VISCOSITY of wafer. The pressure dependence of the effective hydrodynarmc radius for tire BSA system (sohd circles) I$ depicted rn fig. 1. Irut~ally, the BSA moleculesscem to contract slightly, wluch probably indtcates some sort of stabkatlon effect. At pressures above 500 atnl, R H UlCreaSeS P~C~lC~y defray With mcreimg pressure. Over the pressure range studied f?~ increases by --45%. In the previous PCS study [I I] of BSA m acetate buffer an equrvalent tncrease tn RH durmg the denaturatlon process was reported and the observed mmnnlrm in RH was found to be located at &most tfle same pressure. These signs indicate that the pressure dependence of RH 1snot sensttkve to moderate changes of the enwonment. In contrast, a recent heat-scatter study [X!] on the thermal denaturatlon of BSA showed that the change in particle sue during the denaturatton process was correlated to the actual ioruc strength. Furthermore, it 1swell known [1,13,X!,33i tflat thermal denatuntion of a protein is often accomparued by aggregation of the protein molecules, the rate of aggregation is a dxect function of $f and ionic strength. Usually, the aggregation effects are more pronounced the weaker the electrostatic rep&tons between the molecules. Strong aggre gntion effects have also been observed 1341 using an ~trac~ntrtfuge and gel electropfloresism the anaIysis
FIN. 1. Pressure dependence of the effective h~drodymmlc radms for the followmg systems’ l BSA mTrls-buffer (PH = 7 4, I = 0.04 M and e = 4.70 kg m’3];o N@SS (IV = 7.8 X 10’) III 0.1 bl NC! (c = 2 59 kg mW3) 125 J. ‘RR wlucs of conLe~~tlon refer to atmosphertc pressure.
138
10
LCTTCRS
September1982
of BSA solutions which had been exposed to pressures of 1000-3000 atm. However, in the present PCS mvestrgatton, no alarrnmg signs of aggregation could be observed in the data used m the evaluation of RH. viz. there was no unexpected mcreaseof the
scattered mtenstty and the computed normalized variance (Q) m the ~t~bution of ~fusion coefiicients did not increase significantly with pressure. Thus we do not have, in the mte~retation of data, to consider the delicate problem [13] of correlation between aggregation and denaturation phenomena. Since the BSA system has a poiyelectrolytic character it was considered to be of mterest to compare the behaviour of RH for BSA with that for an “ordlnary” flexible synthetic polyele~trolyte l&e sodium poly(styrene sulfonate) (NaPSS), in order to get a conception of whether the observed behavlour of RH for BSA is a common feature associated with aqueous polyelectrolyte systems, in general, under h@ pressure. Therefore, the pressure dependence of R H for the system NaPSS/H20 [25] in presence of salt (1 so.1 M) is also &splayed in fig. 1 (op=n circles). We note that R H for the Nap!% system is ~dependent of pressure over the mterval studred. This is an indication of that the observed pattern of behavrourofRH for BSA reflects changes in molecular d~en~ons due to the denaturation processt rather than a peculiar pressure “effect” associated w1t.hthe general polyelectroiyte character of the system.
plalml
Fig. 2, Pressure dependence of the diffuston coeff’icteflt for BSA for the coucentnttons (kg m-‘): A 73.2 and l 4.70 (values at atmospheric pressure).
Volume 91, number 3
CHEMICAL
PHYSICS LETTERS
In fig. 2 the pressure dependence of the drffusion coeSficient for a low (4.7 kg ms3) and a high (73.2 kg m ) concentration are showed. The common feature of the curves represen+Lingthe two concentrations is that D passes through a maxllllum at moderate pressures, and then above 1000 atm falls off quite rapidly with increasing pressure: the decrease m D is much more pronounced for the bigb concentration. These features are parallel to those observed in the prevlously cited PCS study of BSA in acetate buffer. The broken curves in fig. 2 illustrate the expected behavlour of D if only the pressure dependence of the solvent viscosity is taken mto account. In a recent PCS study [17] on polystyrene in toluene It was shown that the pressure dependence of D (both for Iow and h&b concentrations) was entirely correlated to the change in solvent viscosity with pressure and could be put in the form Dp = D ’ Q’/$, where the superscripts 1 and p denote atmospheric and considered pressure, respectively. It is evldent from fig. 2 that this pressure effect of 11can only partially account for the observed pressure dependence of D, Besides this “viscosity effect”, and the denaturation phenomenon (as docussed above) there are other factors, especially in the case of the high concentration, which may contrlbute to the observed behavlour of D. Since the diffusion process is governed by both hydrodynamic and thermodynamic properties a change in, for instance, the thermodynamic conditions with pressure should affect the pressure dependence of D. Aggregation IS an&her factor which may play a role in the judgement of the tifusion data representing the highest concentration at the h&est pressures. When the pressure was released slowly (20 h to reach atmospheric pressure), signs of irreverslbltity were observed, viz. the values of D monitored successively during the release process were consistently smaller than the correspondmg values obtained during the pressure increase. After that the pressure had attained atmosphenc pressure measurements of D were repeated after another 12 h: withm experimental error, no further change in the value of D was observed. These fmdings for the low-concentration sample may be an indication of that there is a fraction of denaturated protein molecules which do not return to their native form when the pressure is released. It is evident from the results presented here that the protein molecules swell during the denaturation
10 Scptembcr 1982
process; this tendency is probably due to distortion of the delicate balance between hydrogen bonding, electrostatic and hydrophobic interactions which together govern the stabdity of the protein structure. It has been argued [26] that moderate pressures [before denaturation occurs (=lOOO atm)J favour the formation of hydrogen bonds between the solute and the solvent and the disruption of hydrophobic bonds among the solute molecules, above thus pressure these trends are reversed in aqueous solutions.
Acknowledgement Financial support from the Swedish Natural Science Research Councd is gratefully acknowledged.
References
Ill
C. Tanford, Advan Protetn Chem. 23 (1968)
PI
K. Suzukl, Y. M~yosaw3 and C. Suzukt. Arch. Btochem.
121.
Blophys. lOl(l963) 225 Brand& R J. Ohveira and C. Westort, Blochcrnlstry
131J.F.
9 (1970) 1038 Suzuki and Y. Tantgucht, m: Tbe effects of pressure on orgamsms, eds M.A. Slctgh and A G Macdonald (Cambndgc Umv. Press, London, 1972) p. 103.
[41 K. [51 A.
21pp and W. Kauzmann, Blochemtstry 12 (1973)
4217. I’51W. Kauzmann, Proceedings of the 4th lntcrnaliondl Conference on lilgh Pressure, Kyoto (1974). Lt, J.W. Hook, H G. Drickamer and G. Webcr, Blochemlstry 15 (1976) 5571. K. Heremans, Rev. Phys Chem. Japan 50 (1980) 260. P. Nteuwenhuyscn, K. Heremans and J. Clauwaert, Etm-
171T.hl. VI VI [lOI IllI
chcm. Blophys. Acra 606 (1980) 292. K. hluller. H.-D. Liidemann and R. Jaemche, Blophyr Chem. 14 (1981) 101. B. Nystrom and J. Roots, J. Chcm. Phys. to be pubbshcd. Jaemcke. Ber. Bunscnges. Phystk. Chcm. 68 (1964)
1121R.
857.
[I31 R. Jaemcke. J. Polym. SCL Cl6 (1967) 2143. I141 RC. Neuman. W. Kauzmann and A. Zlpp, J. Phys. Chem. 77 (1973)
[ISI
2687.
K. Aokb M. hfurala and K. Huamatsu, AnaL Iliochcm. 59 (1974)
14.
1161S. Ckesson,
S. hlalmrud and B. Lundgrcn, Trans. Faraday
Sot. 66 (1970) 3048. I171 J. Roots and B. Nystrom, Macromolecules 15 (1982)
553. 1181D.E. Koppel, J. Chem.
Phyr 57 (1972) 48 14.
239
Volume 9 I, number 3
CHEMICAL PHYSICS LETTERS
1191 P.N. Pusey, D.E. Koppel, D W. SchaeiLr. R.D. CamermlOtero and S H. Koemg. Blochemlstry 13 (1974) 952. [20] K. Vedam and P. Limsuwan. J. Chem. Phys. 69 (1978) 4762. [3-l] K.E Bctt and J.B. Capp~, Nature 207 (1965) 620. 1221 T. Kamata, Intern. J. BIoI. hlacromolecules 1 (1979) 33. 1231 bl. Nakagakl and Y. Sane, Bull Chem. Sot. Japan 46 (1973) 791
1982
[24] K. Aoki, hf. Tanaka, K. Huamatsu and S. Kaneshma, Rev. Phys. Chem. Japan 36 (1966) 111. [ 251 J. Roots and B. Nystrom, unpubhshed results. 1261 K. Suzuki, Y. hliyosawa, M. Tsucluya and Y. Taniguchb Rev. Phys. Chem. Japan 38 (1968) 63.
ERRATA
T. Zemb and C. Chachaty, Alkyl chain conformatIons III a micellar system from the nuclear spm relaxatlon enhanced by paramagnetic Ions, Chem. Phys. Letters 88 (1982) 68. On page68,last Ime: theelectronic relaxation times of bin’+ and Nl’-+ are inverted. it should read !Oeg and IO-” smsteadof 10wl’ and 10-gs.0npage71, caption to fig. 2 (c) and (d) are mverted.
P.Chandand G.C. Upreti, Electron paramagnetic resonance study ofCuzc-doped ammonium iodide single crystals, Chem. Phys. Letters 88 (1982) 309. On page 312, right-hand column, 3rd paragraphIA I should be 190 f 1 G instead of 90 f 1 G. On page 3 13, lig. 3: the markmgson the Yaxlsshould be 2900, 3 100,330O and 3500 G Instead of 3100,3200,3300 and 3400 G.
240
10 September
CHEMICAL
PHYSICS LETTERS
STUDY OF PRESSURE-INDUCED DENATURATION
10
September
1982
OF BOVlNE SERUM ALBUMIN
BY PHOTON CORRELATION SPECTROSCOPY
B. NYSTROhl l and J ROOTS ** b~srrrrrrcof ~I~~rcalClrmnrsrr~~,Unrwrsrry
of Uppsala. P 0. Bor 532, S-751 -71 Uppsala. Swederr
Rcccircd 10 June 1982, m fin.11form 3 July 1982
The prcssurc-mduced dcnawnrlon al bovine serum albums (BSA) m w-buffer atpH 7.4 was studied m the pressure rang l-4500 alni usrng the photon correlallon specrroscopy technique. The mfluence of pressure on the hydrodynamic radius and on the dtifuslon cocfficwnl was studled
1. introduction The nnllve structure of a protem 1s often stable over a farly wide range of external conditions, but It can be disrupted by sufficiently drastic changes III phys~al or chenucal environment. The process, during which the folded structure of a protem is changed mto the unfolded form, is known as dmmwu~wt~ In the past the denaturation of common protems has been studied using a variety of denaturing agents such as pH, heat, pressure, and chemical denaturants [ 11. There has been a moderate amount of work on the pressure-mduced denaturation of protem molecules in solutlon [2-IO]. Mo$t of these publications have been concerned with the influence of pressure on therm@ dynamic and related quantities, there is a lack of investigations
de&g
with the mfluence of pressure on
transport and dynanuc propertres. In a recent investigation [ 111 we used photon correlauon spectroscopy (PCS) to study the pressureinduced denaturation
of bovme serum albumn
(BSA)
III acetate
buffer with added salt (~OIUC strength I Z= 0. I2 hi) at the lsoelectrlcpH(4.7) and in the pressure interval l-4000 atm. However, there are some factors
* Presentaddress’Depdrtmentof Chemistry,The Uruverslty of Oslo, P.O. Box 1033, Blmdem, Oslo 3. Noruy.
** Presentaddress Chemlal UK.
236
Department of Chemical Engmekng and Technology, ImpcrwlCollege, London SW7 2BY,
which may constitute complications in the analysis of data. Firstly, the employedpH and ionic strength are expected to represent a situation where the electrostatic forces are strongly screened, there are indications III the Merature [ 12,131 that such expenmental conditions favour formation of aggregates in protein solutions. Secondly, it has been shown 1141 that pH of an acetate buffer decreases by approximately one pH unit over a pressure range of 6500 atm. In mew of these aspects, and the knowledge that the features of the denaturation process usually are affected by environmental alteratrons, such aspH and ionic strength, we decided to perform measurements under different and more well-defmed expenmental conditions. The sun of this investigation IS to study how the pressure-induced denaturation affects the size and the diffusion behaviour of the protein molecules. By USIII~ pressure as the denaturant we may learn more about the factors which determme the structure and stablty of protein molecules. In the present work we have studied the pressure denahrrauon of BSA m a MS-buffer (no salt was added) of ionic strength 0.04 M and at pH = 7.4 by means of PCS. At these conditions electrostatic repulsions between the BSA molecules can be expected; possible aggregation effects should be suppressed. In addition, it has been observed [14] that pH of a trisbuffer solution is practically insensitive to pressure in the interval I-6500 atm.
0 009-2614/82/0000-0000/%
02.75 0 1982 North-Holland
Volume 91, number
3
CHEMICAL
PHYSICS LITTERS
IO Scptcmbcr 1987
2. Experimental
gle-mode operation. Light scattered at a scattering
2.1 Matenalr atrd preparatrorl olsolutiorls
angle 0 = 90” was imaged on the photocathode of an In FW130 photomultipher tube. The resulting pulses
A sulfhydryl blocked BSA monomer sample (characterized as 99% monomer) obtained as standard protein powder from hZlles Laboratories (stock no. 81-028-2) was utilized in these experiments. The pH of the protein solutions was adjusted to pH = 7.4 in a t&buffer of ionic strength 0.04 M. BSA concentrations (c) were determined spectrophotometrically using a value for the extmction coefficient, Et?&, of 6.67 mol-’ cm-’ [IS] at 280 MI. The solutions were filtered through 0.22 Mm Mdlipore falters directly mto pre-cleaned hght scattering cuvettes of 16 mm path length in an atmosphere of filtered air. The solutions were kept at 4’C until use and all measurements were taken within four days of preparation of the BSA monomer solutions. 2 2. Eq~t~pmer~tand data analysis The high-pressure vessel has been described in detd elsewhere [ 161. The equipment used in the present PCS measurements was the same as that descriied previously [11,17J. A three-window portable optical high-pressure vessel 1sused, cor.tairtmg the sample solution within a cylindrical quartz cuvette, which has an optically flat bottom and two oppositely placed, optIcally flat wmdows; the cuvette was closed with a movable blackoxidized alummmm plunger, which transmits the pressure and isolates the sample soUlon from the pressure-transmitting fluid glycerol Pressure is applied
with a small hydraulic press, equipped with a Bourdon gauge which has been accurately cahbrated. In ail measurements, the pressure is slowly increased and the sample solution is allowed to equilibrate (2-3 h) before data are accumulated over an interval of l-5 mm. The h&pressure cell is enclosed in an insuiatmg Jacket, through which a water-ethanol mixture is CLTculated from a thermostatted bath. By this arrangement the temperature in the sample cuvette could be maintained constant within +O.l’C. The measurements were carried out at 25’C. A Coherent Radiation model CR4 argon ion laser was used at a wavelength of 488 nm with an etalon frequency stabilizer in the optical cavity to ensure sin-
were amphfied and discriminated and then fed mto a 128.channel dlgitti correlator (Langley-Ford Instruments) wh~h was set to generate tbefiilf autocorrelation function of the scattered mtensity. The correlator was mterfaced to a Luxor ABC 80 microcomputer, which was programmed to calculate the normahzed full-photon counting time correlation function, and to analyse and store data. In tlus study the homodyne configuration was bsed. Intensity correlation data were routinely analysed usmg basically the method of cumulants [18,19] III order to provide the average decay rate r and the normalized variance Q; this latter quantity is a measure of the width of the btribution of the decay rates. It was found that the experimental correlation functions could be well represented by single exponentlals (Q < O.OS),except for those representing the highest concentration at the highest pressures, where increasing departure from “single exponentiahty” was observed. The chffusion coefficient D was determined from the average decay rate r according to i==Dq’,
(1)
where q =(471/A) H sm(O/?,) 1sthe magnitude of the scattering vector, X is the incident light wavelength, II is the index of refraction of solution, and 0 is the scattenng angle. In this study II was determmed from the measured refractive index of solution and the known 1201 pressure dependence of the index of refractlon of WPflx.
3. Results and discussion
Photon cocrelatton spectroscopy constitutes a powerful tool to study diffusion as well as to determine the actual average size change of the protem molecules during the denaturation process. The effective hydrodynamic radius RH may be obtamed from the StokesEmstein relation RH = kT/6nqD ,
(2)
where k is Boltzmann’s constant, T is the absolute temperature, and q is the solvent viscosity. In this work, values of q at tiferent pressures were estunated 237
\‘olume 9 1. nulllber 3
CliCXIiCAl.
PHYSICS
from the vtscosity of the buffer system and the reported [Z I j pressure dependence of the VISCOSITY of wafer. The pressure dependence of the effective hydrodynarmc radius for tire BSA system (sohd circles) I$ depicted rn fig. 1. Irut~ally, the BSA moleculesscem to contract slightly, wluch probably indtcates some sort of stabkatlon effect. At pressures above 500 atnl, R H UlCreaSeS P~C~lC~y defray With mcreimg pressure. Over the pressure range studied f?~ increases by --45%. In the previous PCS study [I I] of BSA m acetate buffer an equrvalent tncrease tn RH durmg the denaturatlon process was reported and the observed mmnnlrm in RH was found to be located at &most tfle same pressure. These signs indicate that the pressure dependence of RH 1snot sensttkve to moderate changes of the enwonment. In contrast, a recent heat-scatter study [X!] on the thermal denaturatlon of BSA showed that the change in particle sue during the denaturatton process was correlated to the actual ioruc strength. Furthermore, it 1swell known [1,13,X!,33i tflat thermal denatuntion of a protein is often accomparued by aggregation of the protein molecules, the rate of aggregation is a dxect function of $f and ionic strength. Usually, the aggregation effects are more pronounced the weaker the electrostatic rep&tons between the molecules. Strong aggre gntion effects have also been observed 1341 using an ~trac~ntrtfuge and gel electropfloresism the anaIysis
FIN. 1. Pressure dependence of the effective h~drodymmlc radms for the followmg systems’ l BSA mTrls-buffer (PH = 7 4, I = 0.04 M and e = 4.70 kg m’3];o N@SS (IV = 7.8 X 10’) III 0.1 bl NC! (c = 2 59 kg mW3) 125 J. ‘RR wlucs of conLe~~tlon refer to atmosphertc pressure.
138
10
LCTTCRS
September1982
of BSA solutions which had been exposed to pressures of 1000-3000 atm. However, in the present PCS mvestrgatton, no alarrnmg signs of aggregation could be observed in the data used m the evaluation of RH. viz. there was no unexpected mcreaseof the
scattered mtenstty and the computed normalized variance (Q) m the ~t~bution of ~fusion coefiicients did not increase significantly with pressure. Thus we do not have, in the mte~retation of data, to consider the delicate problem [13] of correlation between aggregation and denaturation phenomena. Since the BSA system has a poiyelectrolytic character it was considered to be of mterest to compare the behaviour of RH for BSA with that for an “ordlnary” flexible synthetic polyele~trolyte l&e sodium poly(styrene sulfonate) (NaPSS), in order to get a conception of whether the observed behavlour of RH for BSA is a common feature associated with aqueous polyelectrolyte systems, in general, under h@ pressure. Therefore, the pressure dependence of R H for the system NaPSS/H20 [25] in presence of salt (1 so.1 M) is also &splayed in fig. 1 (op=n circles). We note that R H for the Nap!% system is ~dependent of pressure over the mterval studred. This is an indication of that the observed pattern of behavrourofRH for BSA reflects changes in molecular d~en~ons due to the denaturation processt rather than a peculiar pressure “effect” associated w1t.hthe general polyelectroiyte character of the system.
plalml
Fig. 2, Pressure dependence of the diffuston coeff’icteflt for BSA for the coucentnttons (kg m-‘): A 73.2 and l 4.70 (values at atmospheric pressure).
Volume 91, number 3
CHEMICAL
PHYSICS LETTERS
In fig. 2 the pressure dependence of the drffusion coeSficient for a low (4.7 kg ms3) and a high (73.2 kg m ) concentration are showed. The common feature of the curves represen+Lingthe two concentrations is that D passes through a maxllllum at moderate pressures, and then above 1000 atm falls off quite rapidly with increasing pressure: the decrease m D is much more pronounced for the bigb concentration. These features are parallel to those observed in the prevlously cited PCS study of BSA in acetate buffer. The broken curves in fig. 2 illustrate the expected behavlour of D if only the pressure dependence of the solvent viscosity is taken mto account. In a recent PCS study [17] on polystyrene in toluene It was shown that the pressure dependence of D (both for Iow and h&b concentrations) was entirely correlated to the change in solvent viscosity with pressure and could be put in the form Dp = D ’ Q’/$, where the superscripts 1 and p denote atmospheric and considered pressure, respectively. It is evldent from fig. 2 that this pressure effect of 11can only partially account for the observed pressure dependence of D, Besides this “viscosity effect”, and the denaturation phenomenon (as docussed above) there are other factors, especially in the case of the high concentration, which may contrlbute to the observed behavlour of D. Since the diffusion process is governed by both hydrodynamic and thermodynamic properties a change in, for instance, the thermodynamic conditions with pressure should affect the pressure dependence of D. Aggregation IS an&her factor which may play a role in the judgement of the tifusion data representing the highest concentration at the h&est pressures. When the pressure was released slowly (20 h to reach atmospheric pressure), signs of irreverslbltity were observed, viz. the values of D monitored successively during the release process were consistently smaller than the correspondmg values obtained during the pressure increase. After that the pressure had attained atmosphenc pressure measurements of D were repeated after another 12 h: withm experimental error, no further change in the value of D was observed. These fmdings for the low-concentration sample may be an indication of that there is a fraction of denaturated protein molecules which do not return to their native form when the pressure is released. It is evident from the results presented here that the protein molecules swell during the denaturation
10 Scptembcr 1982
process; this tendency is probably due to distortion of the delicate balance between hydrogen bonding, electrostatic and hydrophobic interactions which together govern the stabdity of the protein structure. It has been argued [26] that moderate pressures [before denaturation occurs (=lOOO atm)J favour the formation of hydrogen bonds between the solute and the solvent and the disruption of hydrophobic bonds among the solute molecules, above thus pressure these trends are reversed in aqueous solutions.
Acknowledgement Financial support from the Swedish Natural Science Research Councd is gratefully acknowledged.
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ERRATA
T. Zemb and C. Chachaty, Alkyl chain conformatIons III a micellar system from the nuclear spm relaxatlon enhanced by paramagnetic Ions, Chem. Phys. Letters 88 (1982) 68. On page68,last Ime: theelectronic relaxation times of bin’+ and Nl’-+ are inverted. it should read !Oeg and IO-” smsteadof 10wl’ and 10-gs.0npage71, caption to fig. 2 (c) and (d) are mverted.
P.Chandand G.C. Upreti, Electron paramagnetic resonance study ofCuzc-doped ammonium iodide single crystals, Chem. Phys. Letters 88 (1982) 309. On page 312, right-hand column, 3rd paragraphIA I should be 190 f 1 G instead of 90 f 1 G. On page 3 13, lig. 3: the markmgson the Yaxlsshould be 2900, 3 100,330O and 3500 G Instead of 3100,3200,3300 and 3400 G.
240
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