Search for content and authors

Pressure-Assisted Cold Denaturation of Proteins Compared to Pressure and Heat Denaturation. Application to Food Components

Laszlo Smeller 2Karel Heremans 1Filip Meersman 1

1. K. U. Leuven, Dept. of Chemistry and Biological Dynamics, 200 D CelestijnenlaanB-3001, Leuven 3001, Belgium
2. Semmelweis University of Medicine, Institute of Biophysics and Radiation Biology, Budapest, Hungary


I [Image43.gif]
Internet Journal of the High Pressure School
Proceedings of the III International Warsaw, 13-16 Sept. 1999. Edited
by w. Lojkowski

No part of this text can be reproduced without the written permission
of the authors.
Published with the permission of the authors of the article


Filip Meersman1, Laszlo Smeller2 & Karel Heremans1
Katholieke Universiteit Leuven, Department of Chemistry,
Celestijnenlaan 200D, B-3001 Leuven, Belgium
Institute of Biophysics and Radiation Biology, Semmelweis University
of Medicine, H-1444 Budapest, Hungary

Knowledge of the structure of the cold denatured state of proteins
might be of importance due to the fact that for several proteins it
resembles the structure of an early folding intermediate [1]. Thus it
could be useful for our understanding of protein folding. Another
interesting point is the discussion whether the cold, heat and
pressure denatured states are conformationally and thermodynamically
equivalent or not. We also investigated the aggregation tendency of
the denatured states which is of growing interest since certain
molecular diseases are known to be due to aggregation [2].
Materials and Methods
Horse heart metmyoglobin was obtained from Sigma and dissolved
(75mg/ml) in 10mM Tris-DCl buffer, pD 8.2. The sample was stored
overnight to ensure a sufficient H/D-exchange. For both the cold and
pressure denaturation we used the diamond anvil cell (Diacell
Products, Leicester, UK). A small amount of BaSO[4] was added as an
internal pressure standard [3]. The cold denaturation experiments were
pressure-assisted. A pressure of approximately 200 MPa was applied to
allow measurements down to -25 oC without freezing the protein
solution. For the heat experiment a CaF[2] cell and a Graseby Specac
Automatic Temperature Controller were used. The infrared spectra were
obtained with a Bruker IFS66 FTIR spectrometer equipped with a liquid
nitrogen cooled broad band MCT solid state detector. 250
interferograms were coadded after registration at a resolution of 2
Results and Discussion
The native state of metmyoglobin is for over 75% made out of a-helix
structure [4]. The remaining 20-25% consists of extended chain and
turn structure. The cold and pressure denatured states are
characterised by a persistent amount of secondary structure. The heat
denatured state differs from the former two by the formation of two
bands at 1616 cm-1 and 1683 cm-1, respectively. The appearance of
these two bands is due to intermolecular antiparallel b-sheet
aggregation [5]. However, metmyoglobin does not contain any b-sheet
structure. A hypothetical explanation for this phenomenon would be the
formation of b-sheet out of a-helix during the denaturation process.
The loss of certain helix stabilizing contacts would cause this
transformation, whereby the b-sheet structure is the more
energetically stable structure under the given conditions. This would
also explain the fact that a molecule that will aggregate is not
completely denatured but still has some secondary structure. Moreover
we found that the structure of this pre-aggregated state is similar to
the structure of the cold and pressure denatured states.
Another observation is that without the pressure assistance we cannot
observe a cold denaturation transition before the formation of ice.
This might suggest that pressure shifts the transition to higher
temperatures or the role of pressure in this process is
underestimated. The latter has also been suggested by others [6]. A
possible mechanism to explain the pressure denaturation is that
pressure squeezes the water into the protein and thereby destabilizes
it [7]. Thus it is not unlikely that a pressure of 200 MPa is
responsible, at least in part, for the observed cold denaturation.
This brings us to a final aspect of the denaturation, which is the
role of the solvent. Klotz has pointed at the parallel between protein
behaviour and the anoumalous behaviour of water as a function of
temperature [8]. In case of the cold denaturation experiments we
observed a correlation between the change of the water structure and
the denaturation. The midpoints of the plots of the intensities of the
water band at 3400 cm-1 and the 1566 cm-1 band versus decreasing
temperature coincide with the midpoint of the maximum amide I' band
transition, which is indicative of the secondary structure of the
protein. The changes of the 1566 cm-1 band are attributed to the
solvation of the COO-
of the glutamate side chains [5].
It is clear that the contributions of the solvent and, in case of the
pressure-assisted cold denaturation, the pressure are still not well
understood. The anoumalous behaviour of water that can be observed
with temperature disappears at high pressure [9]. So the
pressure-induced denaturation cannot simply be interpreted in terms of
changes in the water structure.
[Image44.gif] In a more food related topic we investigated the
pressure-induced phase separation of ternary mixtures consisting of a
protein, a polysaccharide and water. For this purpose we used an IR
microscope that enables us to focus on one particular phase and to
determine, spectrally, its composition. This is shown in Figure 1. It
was found that the onset of the phase separation is determined by the
denaturation of the protein.

Figure 1 :
FTIR microscopy of a ternary mixture of bovine serum albumin (BSA)
with methylcellulose (MC) in water. Pure methylcellose (A),
unseparated mixture (B), MC rich phase (C) and the BSA rich phase (D).

The results presented here were obtained with the support of the
Research Fund of the K.U.Leuven, F.W.O. Flanders, Belgium and the
European Community.

[1] Nash, D.P. & Jonas, J., Biochem. Biophys. Res. Comm., 238, 289-291
[2] Booth et al., Nature, 385, 787-793 (1997)
[3] Wong, P.T.T. & Moffat, D.J., Appl. Spectr., 43, 1279-1281 (1989)
[4] Evans, S.V. & Brayer, G.D., J. Mol. Biol., 213, 885-897 (1990)
[5] Ismail, A.A., Mantsch, H.H. & Wong, P.T.T., Biochim. Biophys.
Acta, 1121, 183-188 (1992)
[6] Tsuda, S., Miura, A., Gagné, S.M., Spyracopoulos, L. & Sykes,
B.D., Biochemistry, 38, 5693-5700 (1999)
[7] Wroblowski, B., Diaz, J.F., Heremans, K. & Engelborghs, Y.,
Proteins : Struct., Funct. Gen. 25, 446-455 (1996)
[8] Klotz, I.M., J. Phys. Chem. B, 103, 5910-5916 (1999)
[9] Eisenberg, D. & Kauzmann, W. (1969), The Structure and
Properties of Water, Clarendon Press, Oxford
No part of this text can be reproduced without the written permission
of the authors.
Published with the permission of the authors of the article


Legal notice
  • Legal notice:

Related papers

Presentation: poster at High Pressure School 1999 (3rd), by Filip Meersman
See On-line Journal of High Pressure School 1999 (3rd)

Submitted: 2003-02-16 17:33
Revised:   2009-06-08 12:55