PAC Studies of BSA Conformational Change
Hyperfine Interactions (2004) 159:323–329
DOI 10.1007/s10751-005-9122-3
#
Springer 2005
PAC Studies of BSA Conformational Changes
=
J. GRYBOŚ1,*, M. MARSZAL
EK1, M. LEKKA1, F. HEINRICH2
2
and W. TRÖGER
1
The Henryk Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences,
Radzikowskiego 152, 31-342 Kraków, Poland; e-mail: [email protected]
2
Institute of Experimental Physics II, University of Leipzig, Linnéstraße 5, 04103 Leipzig
Germany
Abstract. The structure of biological molecules plays an important role in many biological
processes. The variation of ambient parameters such as pH, temperature or pressure can influence
important properties of protein molecules like conformation or stability. By means of the perturbed
angular correlation (PAC) technique and atomic force microscopy (AFM) a conformation change
of bovine serum albumin (BSA) molecules has been studied as a function of the pH value of the
BSA solution. The observed decrease of the rotational correlation time and the increase of
molecule diameter as a function of pH were attributed to conformational changes of bovine serum
albumin induced by different pH values of the BSA solution.
Key Words: albumin conformation, atomic force microscopy, AFM, dynamic hyperfine
interaction, nuclear quadrupole interaction, NQI, rotational correlation time, perturbed angular
correlation of g-rays, PAC.
1. Introduction
Bovine serum albumin is one of the most abundant proteins in blood circulatory
systems. They provide many functions like, e.g., maintaining osmotic pressure or
pH. Albumins participate in the transport of a variety of ligands whose affinity
depend on the state of the protein which is affected by the pH and the calcium
concentration in the blood. Since these conditions vary in different tissues and
organs, the characterization of albumin isoforms may help for a better
understanding of its functionality [1].
The primary structure of BSA is constituted from a single polypeptide chain
of 582 amino acid residues and its amino acid sequence is well known [2]. Its
secondary structure, in the native form, is formed by 67% of a-helixes with six
turns, 33% of b-sheets, and 17 disulfide bridges. The protein was modeled as an
ellipsoid with axes of 14 nm 4 nm, with three domains in line.
*Author for correspondence.
324
J. GRYBOŚ ET AL.
It is known that albumin undergoes pH dependent conformational changes
in alkaline and acidic solutions [3]. These conditions influence the forms of
the BSA molecules and they are classified as expanded (E, below pH 2.7),
fast (F, below pH 4.3), native (N, pH 7.0), basic (B, above pH 8.0) and
aged (A, above pH 10.0). These five forms are characterized by a different
content of a-helixes, b-sheets and random coils [4]. At a pH value between 2.7
and 4.3, albumin is in the fast form which is characterized by an increase of the
intrinsic viscosity, and a significant loss of the helical content is observed [5].
The native form with a globular shape is predominant in the pH range 4.5Y7.0
[3].
In this paper, two different techniques, perturbed angular correlation of g-rays
and atomic force microscopy, were applied to study the size and dynamics of
BSA molecules in aqueous solutions at different pH. By AFM the diameters, by
PAC the rotational correlation times of the BSA molecules were determined.
Both parameters depend on pH.
2. Samples preparation and experiment
The BSA molecules for the AFM imaging were immobilized on glass coverslips
activated with a 2.5% glutaraldehyde (supplied by Sigma) aqueous solution for
30 min. Before activation, they were silanized in 4% solution of 3-aminopropyltriethoxysilane (APTES, Sigma) in toluene. The protein immobilization
was done by substrate immersion in 0.5 mg/ml aqueous protein solution for one
hour.
For the PAC experiments a radioisotope, the PAC probe, has to be attached to
the BSA molecule. Here, we used the PAC probe 111In(EC)111Cd, which decays
from 111In to 111Cd via an electron capture (EC) before the g-g-cascade occurs
which is used for the PAC experiments. The samples for the PAC experiments
were prepared in the following way: first, a 200 mM aqueous solution of the
bovine serum albumin (Sigma, Mw = 69 kDa) was prepared which was incubated
with a solution of 111InCl3 (no carrier added) at 30-C for 1 h in order to assure
better binding of the In3+ ions. The sample activity was in the range of 100 to
400 kBq. The total sample volume was about 1Y2 ml. The pH value of the BSA
solution varied from 2 to 10 and it was checked before and after each
measurement. PAC experiments were performed at room temperature and at
liquid nitrogen temperature.
The AFM images were recorded using silicon nitride cantilevers with a spring
constant of 0.03 N/m and the radius of curvature was about 50 nm. The
measurements were carried out at room temperature in deionized water using a
Bliquid cell^ setup. The pH value of the solution was adjusted to 2.0, 3.5, 5.0, 7.0
and 10.0. The size of the albumin molecules can be calculated from the
dependence [6] between the measured diameter S of the investigated structures
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
325
(assumed to be circular) and their real diameter D, taking into account the radius
R of curvature of the AFM tip:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
D2
S ¼ 2 RD þ
4
ð1Þ
The angular correlations of the 172Y245 keV g-ray cascade in the PAC probe
In(EC)111Cd were measured using a 6 detector PAC spectrometer equipped
with 6 BaF2 detectors [7]. Thirty coincidence spectra Ni(Q,t) (i=1,. . .,30) were
collected simultaneously at angles Q = 90- and 180- between the detectors. For a
detailed description of the data analysis see [8].
In the case of a static nuclear quadrupole interaction the data were fitted by
22
(t):
the perturbation function Gstatic
111
static
ðt Þ
G22
¼
3
X
s2n ðÞcosð!n ðÞtÞexpð!n ðÞtÞ
ð2Þ
n¼0
where s2n are the amplitude coefficients and wn are the frequencies, which are
functions of the asymmetry parameter h=(VxxjVyy)/Vzz of the electric field
gradient (tensor components Vii (i=x,y,z)). dwn is the half width at half-maximum
of the Lorentzian frequency distribution around the mean value wn. Such
distributions arise due to variations of the charge environment of the probe
nuclei. This is typical for frozen solutions, where the EFG is determined
not only by the regular charge distribution in the molecule, but also by
random arrangements of the solvent molecules.
In the case of molecules dissolved in a liquid a relaxation effect is produced
by the rotational diffusion of the molecules. This can be described by the
following perturbation function:
static
ðtÞ
G22 ðtÞ ¼ els t G22
ð3Þ
with the slow relaxation constant ls ¼ t1 and with the assumption that wnt s > 1.
s
In the case of a very rapid molecular motion which corresponds to the condition
wnt s < 1, the perturbation function G22(t) becomes in accordance with the
Abragam and Pound theory [9] a simple exponential form:
G22 ðtÞ ¼ elf t
ð4Þ
where lf is the fast relaxation constant. For the nuclear spin I ¼ 52 of the used
PAC probe 111In(EC)111Cd, lf becomes 1f 100:8!Q2 tc , where t c is the
rotational correlation time [10] describing the mobility of molecules in a
solution, 5Q being the nuclear quadrupole frequency.
326
J. GRYBOŚ ET AL.
Figure 1. The PAC spectra of the
temperature.
111
In-BSA complex at different pH measured at room
Table I. The hyperfine parameters for
111
In-BSA samples at different pH value
pH
f0 [%]
f1 [%]
ls [nsj1]
t s [ns]
f2 [%]
lf [nsj1]
t f [ns]
2.0
3.5
5
7
10
34(7)
33(11)
38(1)
32(1)
35(0)
58(1)
26(11)
16(1)
9(1)
0(0)
0.001(0)
0.001(1)
0.008(1)
0.013(1)
0(0)
925(35)
909(495)
122(13)
77(10)
0(0)
8(1)
41(1)
46(1)
59(1)
65(1)
0.044(10)
0.102(3)
0.124(5)
0.202(10)
0.077(1)
0.9(5)
2.1(5)
3(1)
4(2)
1.6(2)
3. Results and discussion
The PAC spectra recorded at room temperature and at different pH values are
shown in Figure 1. The spectra were fitted with a linear combination of formulae
(3) and (4) and an unperturbed, time independent PAC signal (a0):
static
G22;total ðtÞ ¼ a0 þ a1 e1s t G22
ðtÞ þ a2 e1f t
ð5Þ
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
Figure 2. The PAC spectrum of the
111
327
In-BSA complex at pH 7 measured at 77 K.
Figure 3. The AFM images of BSA molecules measured for three pH: (a) 3.5, (b) 5.0, (c) 7.0.
(Image size 427 nm 427 nm).
The amplitudes a0, a1 and a2 were treated as free parameters. These amplitudes
contain the anisotropy of the cascade, the solid angle correction factors, and the
population of the different states by the PAC probe. In order to compare the
fi ¼
ai
2
P
were calculated and shown together
populations directly the fractions
a
with the hyperfine parameters obtained from the fitting procedure in Table I. The
fraction f1 of the slowly rotating species was assigned to In3+ ions bound to BSA
molecules whereas the fraction f2 with the fast rotating species was attributed to
In3+ ions bound to hydroxyl groups or other small molecules. The unperturbed
fraction f0, which changed only little with pH, was assigned to unbound probe
ions in the solution. In order to determine the fast rotational correlation time t c, it
is necessary to know the parameters of the static nuclear quadrupole interaction.
These parameters were determined from PAC measurements at the liquid
nitrogen and were very similar for all pH values: quadrupole frequency wQ $
22Y23 Mrad/s, asymmetry parameter h $ 0.5 and a large Lorentzian frequency
distribution. The static PAC spectrum of the 111In-BSA complex at pH 7
measured at 77 K is shown in Figure 2. The rotational correlation time of slow
molecular motion is simply given as a reciprocal of the ls value. The values of
both rotational correlation times are also shown in Table I.
The decrease of the fraction of slow relaxation ( f1, ls) accompanied by the
increase of the number of indium ions bound to hydroxyl groups ( f2, lf) with
i
i¼0
328
J. GRYBOŚ ET AL.
Figure 4. The BSA diameter and the rotational correlation time as a function of pH of the
BSA solution.
increasing pH can be explained by conformational changes of the BSA
molecules. The only amino acid of BSA which can bind In, cysteine 34, is located
in the center of the elongated BSA structure, and for the F-conformation of
albumin this binding site is easily accessible for the indium probe. However, an
increase of the pH leads to a conformational change of BSA into the more oval
structure (N), which results in a reduced accessibility of the cysteine 34 for In3+
ions. Such a structural change is also reflected by the rotational time t f which
decreases with the increase of pH. For pH 10, the parameters f1, ls and t s, indicate that the indium ions did not bind to the BSA molecule at all. Previously
reported rotational correlation times for 111In-BSA system were different from
those obtained here [11]. The discrepancy can be explained by the different conditions of the PAC experiments (shorter time scale and less statistical quality of
the PAC spectra of [11]) and by different procedures of the sample preparation.
The second fraction f2 represents 111In3+ ions bound to hydroxyl groups or
other smaller molecules. These smaller molecules may result from the electron
capture after-effects accompanying the radioactive decay of 111In to 111Cd
leading to a partial or entire disintegration of the BSA molecules. This fraction f2
increases with pH at the expense of the fraction f1 and becomes dominant for pH
values larger than 5 since the amount of hydroxyl groups in the solution
increases with increasing pH.
The AFM images of albumin molecules immobilized on a glass surface
for three chosen pH values are presented in Figure 3. The average value of
the diameter of a single albumin molecule was determined by fitting Gaussian
distributions to the histograms of the molecule diameters. Taking into account
the effect of a topography convolution with AFM tip (Equation 1), the real
diameter of the BSA molecule is 10 T 2.5 nm, 11 T 4 nm, 13 T 6 nm and 22 T 5
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
329
nm for pH 2.0, 3.5, 5.0 and 7.0, respectively. It was observed that the molecule
diameter increases as a function of pH. Figure 4 shows the comparison of the
BSA molecule diameter and rotational correlation times.
These results obtained by two different methods confirmed that albumin
molecules undergo conformational changes at different pH value. These structural properties were reflected by the decrease of the rotational correlation time
(PAC) and the increase of the molecule diameter seen in AFM images.
Acknowledgments
The authors acknowledge the support of a PolishYGerman Scientific and
Technical Collaboration (WTZ No 4440/2002) from the Bundesministerium für
Bildung und Forschung and State Committee for Scientific Research.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Carter D. C. and Ho J. X., Adv. Protein Chem. 45 (1994), 153.
Burova T. V., Grinberg N. V., Golubeva I. A., Mashkevich A. Y., Grinberg V. Y. and
Tolstoguzov V. B., Food Hydrocoll. 13 (1999), 7.
Harmsen B. J. M. and Braam W. J. M., Int. J. Protein Res. 1 (1969), 225.
Reed R. G., Feldhoff R. C., Clute O. L. and Peters T. Jr., Biochemistry 14 (1975), 4578.
Harrington W. F., Johnson P. and Ottewill R. H., Biochem. J. 62 (1956), 569.
Engel A., Schoenenberger C. A. and Müller D. J., Curr. Opin. Struct. Biol. 7 (1997), 279.
Butz T., Saibene S., Fraenzke T. and Weber M., Nucl. Instrum. Methods A 284 (1989), 417.
Butz T. Z., Naturforsch. 51a (1996), 396.
Abragam A. and Pound R. V., Phys. Rev. 92 (1953), 943.
Marshall A. G. and Meares C. F., J. Chem. Phys. 56 (1972), 1226.
Martin P. W. and Kalfas C. A., Nucl. Instrum. Methods 171 (1980), 603.
DOI 10.1007/s10751-005-9122-3
#
Springer 2005
PAC Studies of BSA Conformational Changes
=
J. GRYBOŚ1,*, M. MARSZAL
EK1, M. LEKKA1, F. HEINRICH2
2
and W. TRÖGER
1
The Henryk Niewodniczański Institute of Nuclear Physics Polish Academy of Sciences,
Radzikowskiego 152, 31-342 Kraków, Poland; e-mail: [email protected]
2
Institute of Experimental Physics II, University of Leipzig, Linnéstraße 5, 04103 Leipzig
Germany
Abstract. The structure of biological molecules plays an important role in many biological
processes. The variation of ambient parameters such as pH, temperature or pressure can influence
important properties of protein molecules like conformation or stability. By means of the perturbed
angular correlation (PAC) technique and atomic force microscopy (AFM) a conformation change
of bovine serum albumin (BSA) molecules has been studied as a function of the pH value of the
BSA solution. The observed decrease of the rotational correlation time and the increase of
molecule diameter as a function of pH were attributed to conformational changes of bovine serum
albumin induced by different pH values of the BSA solution.
Key Words: albumin conformation, atomic force microscopy, AFM, dynamic hyperfine
interaction, nuclear quadrupole interaction, NQI, rotational correlation time, perturbed angular
correlation of g-rays, PAC.
1. Introduction
Bovine serum albumin is one of the most abundant proteins in blood circulatory
systems. They provide many functions like, e.g., maintaining osmotic pressure or
pH. Albumins participate in the transport of a variety of ligands whose affinity
depend on the state of the protein which is affected by the pH and the calcium
concentration in the blood. Since these conditions vary in different tissues and
organs, the characterization of albumin isoforms may help for a better
understanding of its functionality [1].
The primary structure of BSA is constituted from a single polypeptide chain
of 582 amino acid residues and its amino acid sequence is well known [2]. Its
secondary structure, in the native form, is formed by 67% of a-helixes with six
turns, 33% of b-sheets, and 17 disulfide bridges. The protein was modeled as an
ellipsoid with axes of 14 nm 4 nm, with three domains in line.
*Author for correspondence.
324
J. GRYBOŚ ET AL.
It is known that albumin undergoes pH dependent conformational changes
in alkaline and acidic solutions [3]. These conditions influence the forms of
the BSA molecules and they are classified as expanded (E, below pH 2.7),
fast (F, below pH 4.3), native (N, pH 7.0), basic (B, above pH 8.0) and
aged (A, above pH 10.0). These five forms are characterized by a different
content of a-helixes, b-sheets and random coils [4]. At a pH value between 2.7
and 4.3, albumin is in the fast form which is characterized by an increase of the
intrinsic viscosity, and a significant loss of the helical content is observed [5].
The native form with a globular shape is predominant in the pH range 4.5Y7.0
[3].
In this paper, two different techniques, perturbed angular correlation of g-rays
and atomic force microscopy, were applied to study the size and dynamics of
BSA molecules in aqueous solutions at different pH. By AFM the diameters, by
PAC the rotational correlation times of the BSA molecules were determined.
Both parameters depend on pH.
2. Samples preparation and experiment
The BSA molecules for the AFM imaging were immobilized on glass coverslips
activated with a 2.5% glutaraldehyde (supplied by Sigma) aqueous solution for
30 min. Before activation, they were silanized in 4% solution of 3-aminopropyltriethoxysilane (APTES, Sigma) in toluene. The protein immobilization
was done by substrate immersion in 0.5 mg/ml aqueous protein solution for one
hour.
For the PAC experiments a radioisotope, the PAC probe, has to be attached to
the BSA molecule. Here, we used the PAC probe 111In(EC)111Cd, which decays
from 111In to 111Cd via an electron capture (EC) before the g-g-cascade occurs
which is used for the PAC experiments. The samples for the PAC experiments
were prepared in the following way: first, a 200 mM aqueous solution of the
bovine serum albumin (Sigma, Mw = 69 kDa) was prepared which was incubated
with a solution of 111InCl3 (no carrier added) at 30-C for 1 h in order to assure
better binding of the In3+ ions. The sample activity was in the range of 100 to
400 kBq. The total sample volume was about 1Y2 ml. The pH value of the BSA
solution varied from 2 to 10 and it was checked before and after each
measurement. PAC experiments were performed at room temperature and at
liquid nitrogen temperature.
The AFM images were recorded using silicon nitride cantilevers with a spring
constant of 0.03 N/m and the radius of curvature was about 50 nm. The
measurements were carried out at room temperature in deionized water using a
Bliquid cell^ setup. The pH value of the solution was adjusted to 2.0, 3.5, 5.0, 7.0
and 10.0. The size of the albumin molecules can be calculated from the
dependence [6] between the measured diameter S of the investigated structures
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
325
(assumed to be circular) and their real diameter D, taking into account the radius
R of curvature of the AFM tip:
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
D2
S ¼ 2 RD þ
4
ð1Þ
The angular correlations of the 172Y245 keV g-ray cascade in the PAC probe
In(EC)111Cd were measured using a 6 detector PAC spectrometer equipped
with 6 BaF2 detectors [7]. Thirty coincidence spectra Ni(Q,t) (i=1,. . .,30) were
collected simultaneously at angles Q = 90- and 180- between the detectors. For a
detailed description of the data analysis see [8].
In the case of a static nuclear quadrupole interaction the data were fitted by
22
(t):
the perturbation function Gstatic
111
static
ðt Þ
G22
¼
3
X
s2n ðÞcosð!n ðÞtÞexpð!n ðÞtÞ
ð2Þ
n¼0
where s2n are the amplitude coefficients and wn are the frequencies, which are
functions of the asymmetry parameter h=(VxxjVyy)/Vzz of the electric field
gradient (tensor components Vii (i=x,y,z)). dwn is the half width at half-maximum
of the Lorentzian frequency distribution around the mean value wn. Such
distributions arise due to variations of the charge environment of the probe
nuclei. This is typical for frozen solutions, where the EFG is determined
not only by the regular charge distribution in the molecule, but also by
random arrangements of the solvent molecules.
In the case of molecules dissolved in a liquid a relaxation effect is produced
by the rotational diffusion of the molecules. This can be described by the
following perturbation function:
static
ðtÞ
G22 ðtÞ ¼ els t G22
ð3Þ
with the slow relaxation constant ls ¼ t1 and with the assumption that wnt s > 1.
s
In the case of a very rapid molecular motion which corresponds to the condition
wnt s < 1, the perturbation function G22(t) becomes in accordance with the
Abragam and Pound theory [9] a simple exponential form:
G22 ðtÞ ¼ elf t
ð4Þ
where lf is the fast relaxation constant. For the nuclear spin I ¼ 52 of the used
PAC probe 111In(EC)111Cd, lf becomes 1f 100:8!Q2 tc , where t c is the
rotational correlation time [10] describing the mobility of molecules in a
solution, 5Q being the nuclear quadrupole frequency.
326
J. GRYBOŚ ET AL.
Figure 1. The PAC spectra of the
temperature.
111
In-BSA complex at different pH measured at room
Table I. The hyperfine parameters for
111
In-BSA samples at different pH value
pH
f0 [%]
f1 [%]
ls [nsj1]
t s [ns]
f2 [%]
lf [nsj1]
t f [ns]
2.0
3.5
5
7
10
34(7)
33(11)
38(1)
32(1)
35(0)
58(1)
26(11)
16(1)
9(1)
0(0)
0.001(0)
0.001(1)
0.008(1)
0.013(1)
0(0)
925(35)
909(495)
122(13)
77(10)
0(0)
8(1)
41(1)
46(1)
59(1)
65(1)
0.044(10)
0.102(3)
0.124(5)
0.202(10)
0.077(1)
0.9(5)
2.1(5)
3(1)
4(2)
1.6(2)
3. Results and discussion
The PAC spectra recorded at room temperature and at different pH values are
shown in Figure 1. The spectra were fitted with a linear combination of formulae
(3) and (4) and an unperturbed, time independent PAC signal (a0):
static
G22;total ðtÞ ¼ a0 þ a1 e1s t G22
ðtÞ þ a2 e1f t
ð5Þ
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
Figure 2. The PAC spectrum of the
111
327
In-BSA complex at pH 7 measured at 77 K.
Figure 3. The AFM images of BSA molecules measured for three pH: (a) 3.5, (b) 5.0, (c) 7.0.
(Image size 427 nm 427 nm).
The amplitudes a0, a1 and a2 were treated as free parameters. These amplitudes
contain the anisotropy of the cascade, the solid angle correction factors, and the
population of the different states by the PAC probe. In order to compare the
fi ¼
ai
2
P
were calculated and shown together
populations directly the fractions
a
with the hyperfine parameters obtained from the fitting procedure in Table I. The
fraction f1 of the slowly rotating species was assigned to In3+ ions bound to BSA
molecules whereas the fraction f2 with the fast rotating species was attributed to
In3+ ions bound to hydroxyl groups or other small molecules. The unperturbed
fraction f0, which changed only little with pH, was assigned to unbound probe
ions in the solution. In order to determine the fast rotational correlation time t c, it
is necessary to know the parameters of the static nuclear quadrupole interaction.
These parameters were determined from PAC measurements at the liquid
nitrogen and were very similar for all pH values: quadrupole frequency wQ $
22Y23 Mrad/s, asymmetry parameter h $ 0.5 and a large Lorentzian frequency
distribution. The static PAC spectrum of the 111In-BSA complex at pH 7
measured at 77 K is shown in Figure 2. The rotational correlation time of slow
molecular motion is simply given as a reciprocal of the ls value. The values of
both rotational correlation times are also shown in Table I.
The decrease of the fraction of slow relaxation ( f1, ls) accompanied by the
increase of the number of indium ions bound to hydroxyl groups ( f2, lf) with
i
i¼0
328
J. GRYBOŚ ET AL.
Figure 4. The BSA diameter and the rotational correlation time as a function of pH of the
BSA solution.
increasing pH can be explained by conformational changes of the BSA
molecules. The only amino acid of BSA which can bind In, cysteine 34, is located
in the center of the elongated BSA structure, and for the F-conformation of
albumin this binding site is easily accessible for the indium probe. However, an
increase of the pH leads to a conformational change of BSA into the more oval
structure (N), which results in a reduced accessibility of the cysteine 34 for In3+
ions. Such a structural change is also reflected by the rotational time t f which
decreases with the increase of pH. For pH 10, the parameters f1, ls and t s, indicate that the indium ions did not bind to the BSA molecule at all. Previously
reported rotational correlation times for 111In-BSA system were different from
those obtained here [11]. The discrepancy can be explained by the different conditions of the PAC experiments (shorter time scale and less statistical quality of
the PAC spectra of [11]) and by different procedures of the sample preparation.
The second fraction f2 represents 111In3+ ions bound to hydroxyl groups or
other smaller molecules. These smaller molecules may result from the electron
capture after-effects accompanying the radioactive decay of 111In to 111Cd
leading to a partial or entire disintegration of the BSA molecules. This fraction f2
increases with pH at the expense of the fraction f1 and becomes dominant for pH
values larger than 5 since the amount of hydroxyl groups in the solution
increases with increasing pH.
The AFM images of albumin molecules immobilized on a glass surface
for three chosen pH values are presented in Figure 3. The average value of
the diameter of a single albumin molecule was determined by fitting Gaussian
distributions to the histograms of the molecule diameters. Taking into account
the effect of a topography convolution with AFM tip (Equation 1), the real
diameter of the BSA molecule is 10 T 2.5 nm, 11 T 4 nm, 13 T 6 nm and 22 T 5
PAC STUDIES OF BSA CONFORMATIONAL CHANGES
329
nm for pH 2.0, 3.5, 5.0 and 7.0, respectively. It was observed that the molecule
diameter increases as a function of pH. Figure 4 shows the comparison of the
BSA molecule diameter and rotational correlation times.
These results obtained by two different methods confirmed that albumin
molecules undergo conformational changes at different pH value. These structural properties were reflected by the decrease of the rotational correlation time
(PAC) and the increase of the molecule diameter seen in AFM images.
Acknowledgments
The authors acknowledge the support of a PolishYGerman Scientific and
Technical Collaboration (WTZ No 4440/2002) from the Bundesministerium für
Bildung und Forschung and State Committee for Scientific Research.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
Carter D. C. and Ho J. X., Adv. Protein Chem. 45 (1994), 153.
Burova T. V., Grinberg N. V., Golubeva I. A., Mashkevich A. Y., Grinberg V. Y. and
Tolstoguzov V. B., Food Hydrocoll. 13 (1999), 7.
Harmsen B. J. M. and Braam W. J. M., Int. J. Protein Res. 1 (1969), 225.
Reed R. G., Feldhoff R. C., Clute O. L. and Peters T. Jr., Biochemistry 14 (1975), 4578.
Harrington W. F., Johnson P. and Ottewill R. H., Biochem. J. 62 (1956), 569.
Engel A., Schoenenberger C. A. and Müller D. J., Curr. Opin. Struct. Biol. 7 (1997), 279.
Butz T., Saibene S., Fraenzke T. and Weber M., Nucl. Instrum. Methods A 284 (1989), 417.
Butz T. Z., Naturforsch. 51a (1996), 396.
Abragam A. and Pound R. V., Phys. Rev. 92 (1953), 943.
Marshall A. G. and Meares C. F., J. Chem. Phys. 56 (1972), 1226.
Martin P. W. and Kalfas C. A., Nucl. Instrum. Methods 171 (1980), 603.