A Size-Exclusion Chromatography/FT-IR (SEC/FT-IR) Technique for Improved FTIR Spectroscopy of Proteins in D 2 O Solution

Abstract

A size-exclusion chromatography/Fourier transform infrared spectroscopic (SEC/FT-IR) method for improving the FT-IR spectroscopy of proteins in D₂O solution is described. The technique employs low-volume SEC columns to exchange protein solutions into buffered D₂O and effectively removes spectral interference from H₂O and other low-molecular-weight compounds in the region of the protein amide carbonyl stretching band (1600–1700 cm⁻¹). Application of the method with protein solutions prepared in deuterated, denaturing buffers completely exchanges labile protein hydrogens with deuterium and refolds the proteins into their native conformations for subsequent spectral characterization. Potential applications of the SEC/FT-IR method for kinetic monitoring of dynamic processes in proteins (e.g., secondary structure formation during protein folding) are also discussed.

Keywords

Size-exclusion chromatography Fourier transform infrared spectroscopy

Get full access to this article

View all access options for this article.

References

Susi

, Structure and Stability of Biological Molecules, Timasheff

S. N.

and Fasman

G. D

, Eds. (Marcel Dekker, New York, 1969), pp. 573–633.

Koenig

J. L.

and Tabb

D. L

, in Analytical Applications of FTIR to Molecular and Biological Systems, Durig

J. R.

, Ed. (Reidel, Holland, 1980), pp. 241–255.

Timasheff

S. N.

Susi

and Stevens

, J. Biol. Chem. 242, 5467 (1967).

Suzuki

Iwashita

Shimanouchi

and Tsuboi

, Biopolymers 4, 337 (1966).

Hvidt

and Nielsen

S. O

, Adv. Protein Chem. 21, 287 (1966).

Hellgeth

J. W.

and Taylor

L. T

, J. Chromatogr. Sci. 24, 519 (1986).

White

, Chromatography/Fourier Transform Infrared Spectroscopy and Its Applications (Marcel Dekker, New York, 1990).

Jansen

J. A. J.

, Fresenius J. Anal. Chem. 337, 398 (1990).

Dekmezian

A. H.

Morioka

and Camp

, J. Polym. Sci., Part B: Polym. Phys. 28, 1903 (1990).

10.

Nishikida

Housaki

Morimoto

and Kinoshita

, J. Chromatogr. 517, 209 (1990).

11.

Glasoe

P. K.

and Long

F. A

, J. Phys. Chem. 64, 188 (1960).

12.

Yau

W. W.

Kirkland

J. J.

and Bly

D. D

, Modern Size-Exclusion Chromatography (John Wiley and Sons, New York, 1979), pp. 445–447.

13.

Yau

W. W.

Kirkland

J. J.

and Bly

D. D

, Modern Size-Exclusion Liquid Chromatography (John Wiley and Sons, New York, 1979), pp. 424–426.

14.

Kauppinen

J. K.

Moffat

D. J.

Mantsch

H. H.

and Cameron

D. G

, Appl. Spectrosc. 35, 271 (1981).

15.

Maddams

W. F.

and Southon

M. J

, Spectrochim. Acta 38A, 459 (1982).

16.

Susi

and Byler

D. M

, Biochem. Biophys. Res. Commun. 115, 391 (1983).

17.

Ruegg

Metzger

and Susi

, Biopolymers 14, 1465 (1975).

18.

Eckert

Grosse

Malur

and Repke

K. R. M

, Biopolymers 16, 2549 (1977).

19.

Lee

D. C.

Harris

P. I.

Chapman

and Mitchell

R. C

, Biochemistry 29, 9185 (1990).

20.

Odorzynski

T. W.

and Light

, J. Biol. Chem. 254, 4291 (1979).

21.

Corbett

R. J. T.

and Roche

R. S

, Biochemistry 23, 1888 (1984).

22.

Light

and Higaki

J. N

, Biochemistry 26, 5556 (1987).

23.

Hall

B. T.

, in Protein Folding, Gierasch

L. M.

and King

, Eds. (American Association for the Advancement of Science, Washington, D.C., 1990), pp. 198–207.

24.

Creighton

T. E.

, J. Mol. Biol. 137, 61 (1980).

25.

Kuwajima

Hiraoka

Ikeguchi

and Sugai

, Biochemistry 24, 874 (1985).

26.

Beasty

A. N.

and Matthews

C. R

, Biochemistry 2. 3547 (1985).

27.

Anfinsen

C. B.

, Science 181, 223 (1973).

28.

Creighton

T. E.

, J. Phys. Chem. 89, 2452 (1985).

29.

Creighton

T. E.

, Prog. Biophys. Mol. Biol. 33, 231 (1978).

30.

Light

, BioTechniques 3, 298 (1985).