Prince Eric Perez
580 California St., Suite 400
San Francisco, CA, 94104
Do Denatured Proteins Behave Like Polymers?
1994, Macromolecules
https://doi.org/10.1021/MA00090A042visibility
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Abstract
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This research investigates the behavior of denatured proteins, specifically human serum albumin, in relation to their interactions with surfaces and how they compare to linear polymers. Using surface force measurement techniques, findings demonstrate that denatured human serum albumin behaves similarly to linear polymers when adsorbed onto smooth mica surfaces, governed by interactions between the protein molecules, the surface, and the solvent conditions. The results challenge the notion that denatured proteins exist solely in a linear structure, providing molecular-scale confirmation of their flexible polymer-like behavior.
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3424
Macromolecules 1994,27, 3424-3425
Notes
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Do Denatured Proteins Behave Like P o l y m e r s ?
Frederic Pincet? Eric Perez3 and Georges Belfort'
Bioseparations Research Center and The Howard P .
Isermann Department of Chemical Engineering, Rensselaer
Polytechnic Institute, Troy, New York 12180-3590
Received December 20, 1993
Introduction
Since proteins i n vivo are usually confined to very small
spaces (specific regions of the cell) and since proteins in
vitro are often associated with surfaces (catheters, dialysis
membranes, separation devices such as chromatography
packings and synthetic membrane filters, and marine
surfaces such as hulls), much recent research has been
focused on the folding behavior of proteins adsorbed at
various interfaces and under different conditions.l,*JOJ1
Many different experimental methods have been used in
these studies to probe the kinetic and equilibrium behavior
of adsorbed proteins. Little attention has, however, been
devoted to the behavior of unfolded or denatured proteins.
Although it has been implicitly assumed that unfolded or
denatured proteins are linear in structure, solely dependent
on their covalent amino acid sequence, experimental
evidence confirming this has been wanting. We address
this question here by using methods that have been widely
used for studying adsorbed linear polymers with unfolded
denatured human serum albumin as a model protein.
Experimental Section
Normalized force-distance measurements were obtained for
two experiments with adsorbed human serum albumin (HSA;
molecular weight 66 500, #A 3782, Sigma,St. Louis, MO) in a
M KC1 solution at pH 2 (see the data points in Figure 1). In the
first experiment, HSA was adsorbed onto mica for 2 h from a 5
mg/L protein solution containing
M KC1 at pH 2. After the
adsorption step, the protein-containing solution was replaced
with a
M KC1 solution at the same temperature (20 f 0.5
"C) and the forces were measured as a function of separation.
This solution was then replaced with a 10 M urea and
M KC1
solution at pH 2, and the forces were again measured as a function
of separation. We assume that constrained or restricted equilibrium existed, Le., the protein was irreversibly adsorbed, and
that on replacement of the protein solution with the KC1solution
little if any protein desorbed into the solution. This was
confirmed by the absence of proteins in the KCl solution after
the experiments. The technique involvesthe directmeasurement
in a surface force apparatus of the intermolecular forces between
two cross-mica cylinders onto which HSA has been preadsorbed
for 2 h and uses interferometry and the spring constant to
determine both the separation distance (D)and the forces.6
Dividing the measured force by the radius of curvature of the
mica cylinders ( R = 2 cm) obtains the interactionenergy between
the two surfaces using the Derjaguin appr~ximation.~
Results a n d Discussion
For constrained equilibrium with polymers, the amount
of noninteracting linear polymer molecules between two
mica surfaces is constant and independent of D. Hence,
for small D , where the polymer concentration between
I
0.01
lo
distance (nm)
1o2
Figure 1. Normalized force versus distance of separation for
mica surfaces immersed in a 5 mg/L human serum albumin (HSA)
and le2M KC1solution at 20 f 0.5 O C . Thesolidcirclesrepresent
the force-distance profile at pH 2 with 10 M urea added to the
solution to denature the protein. A linear regression gives aD-lJ6
dependence for the short-rangeforces and a D-2.sdependence for
the long-range forces. The open circles represent the forcedistance profile at pH 2 without urea. A linear regression gives
a D-2 dependence for the forces. At short distance (8.5 nm) the
profile is vertical on a log-log scale. The decay length is 5.6 nm,
and the deposited layer thickness is 4.2 f 0.5 nm. The crosses
represent the force-distance profile at pH 9 without urea. A
linear regression gives a D-3.4dependence for the force-distance
profile. At short distance (8.5 nm) the profile is vertical on a
log-log scale. The decay length is 3.7 nm, and the deposited
layer thickness is 4.3 f 1.0 nm. The lines represent the linear
regression for each case.
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the surfaces is constant, de Gennes3p4has shown that the
free energy of interaction between the surfaces scales with
D-*.25. For large D, the polymer concentration in the
solution is negligible as compared to that adsorbed on the
surfaces, and the free energy of interaction between the
surfaces scales with D2. Note that these exponents are
independent of the number of monomers in the polymer
chains. Since these and other relationships have been
tested for polymer adsorption under constrained equil i b r i ~ mwe
, ~decided
~~
t o use them to determine whether
denatured proteins (under acid transition a t pH 2 and a
combination of low p H with a strong denaturing agent, 10
M urea) also follow these scaling laws.
In the presence of 10 M urea and a t p H 2, HSA behaves
according to the near and far distance scaling laws
predicted for a polymer and is most likely fully unfolded
and denatured (solid circles and lines in the upper curve
in Figure 1). Without urea a t p H 2, the globular protein
HSA undergoes acid transition that produces partially
unfolded states reminiscent of the globular state.' This
could explain the observed D-2.0 dependence at shorter
distances for the far field and the much lower energy of
interaction between the adsorbed molecules (open circles).
The measured decay length on a semilog plot for this case
(without urea) is 5.6 nm which is larger than that expected
for electrostatic interactions of 2.2 nm (Debye decay length)
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* Corresponding author.
Present address: Laboratoire de Physique Statistique, Ecole
Normale Superieure, 24 rue Lhomond, 75005 Paris, France.
t
0024-929719412227-3424$04.5010
0 1994 American Chemical Society
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Notes 3425
Macromolecules, Vol. 27, No. 12, 1994
at pH 2. The deposited layer thickness on one surface
was about 4.2 f 0.5 nm, about the size of the side-on
dimension of 4 nm for HSA. This suggests, in addition
to electrostatic interactions, the existence of weak steric
forces probably resulting from the interaction between
partially denatured HSA. As acontrol, we measured BSA/
HSA interactions without urea in
M KC1 and a t pH
9 (crosses in Figure 1). For this case, the normalized force
showed a D-3.4on separation with a decay length of 3.7 nm
and a deposited layer thickness on one surface of 4.3 f 1.0
nm. The decay length is close to that expected from
classical DLVO theory, while the thickness again suggests
a side-on orientation a t this pH.
Using the surface force apparatus and the scaling rules
of de Gennes, we show that unfolded (denatured) human
serum albumin behaves essentially as a linear polymer
while adsorbed onto molecularly smooth mica in
M
KC1 a t pH 2 in the presence and absence of 10 M urea.
This is the first confirmation on a molecular scale that
unfolded (and adsorbed) protein molecules can indeed
behave as flexible polymer molecules and that the interactions between the adsorbed molecules, the adsorbent,
and the solvent determine their molecular state.
Acknowledgment. The authors thank Ed Uzgiris and
Jeffrey Koehler for comments on the manuscript.
References and Notes
(1) Brash, J . L. Proteins at Interfaces, Physicochemical and
Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.;
ACSSvmoosium Series 343: American Chemical Societv:
Washington, DC, 1987;pp 490-505.
(2) de Costello,L.; Luckham, B. A.;Tadros, Th. F. Colloids Surf.
1988/89,34,301-306.
(3) de Gennes. P.-G. Macromolecules 1981.14. 1637-1644.
(4) de Gennes; P.-G. Macromolecules 1982;15;492-500.
(5) Derjaguin, B. V. Kolloid 2.1934,69,155-164.
(6) Israelachvili,J.N.;Adams, G. E.J.Chem. Soc.,Faraday Trans.
1 1987,74,975-1001.
(7) Kuwajima, K. Proteins: Struct., Funct., Genet. 1989,6,87103.
(8) Lee, C.-S.; Belfort, G. Proc. Natl. A c Q ~Sci.
. 1989,86,83928396.
(9)Taunton,H. J.;Toprakcioglu,C.; Fetters,L. J.; Klein,J.Nature
1988,332,712-714.
(10) Wattenbarger, M.R.;Chan, H. S.;Evans, D. F.; Bloomfield,
V. A.; Dill, K. A. J. Chem. Phys. 1990,93 (ll),8343-8351.
(11) Yoon, B. Y.; Lenhoff, A. M. J.Phys. Chem. 1992,96,31303134.
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References (12)
- Brash, J . L. Proteins at Interfaces, Physicochemical and Biochemical Studies; Brash, J. L., Horbett, T. A., Eds.; ACSSvmoosium Series 343: American Chemical Societv: Washington, DC, 1987; pp 490-505.
- de Costello, L.; Luckham, B. A.; Tadros, Th. F. Colloids Surf.
- de Gennes. P.-G. Macromolecules 1981.14. 1637-1644. 1988/89,34,301-306.
- de Gennes; P.-G. Macromolecules 1982; 15; 492-500.
- Derjaguin, B. V. Kolloid 2. 1934, 69, 155-164.
- Israelachvili, J. N.; Adams, G. E.J. Chem. Soc.,Faraday Trans.
- Kuwajima, K. Proteins: Struct., Funct., Genet. 1989, 6, 87-
- Lee, C.-S.; Belfort, G. Proc. Natl. A c Q ~. Sci. 1989,86,8392-
- Taunton, H. J.;Toprakcioglu, C.; Fetters, L. J.; Klein, J.Nature
- Wattenbarger, M. R.; Chan, H. S.; Evans, D. F.; Bloomfield,
- Yoon, B. Y.; Lenhoff, A. M. J. Phys. Chem. 1992,96,3130- 1 1987, 74,975-1001. 103. 8396. 1988,332,712-714.
- V. A.; Dill, K. A. J. Chem. Phys. 1990,93 (ll), 8343-8351. 3134.