A method is described for the quantitative subtraction of water from the transmission infrared spectra of aqueous solutions of proteins. The 2125-cm−1 association band of water is used as an internal intensity standard, and the scaling factor is determined with the use of a second-order least-squares fit. This method eliminates the user bias encountered with interactive methods and takes into account small baseline variations due to instrument drift. Statistical analysis of the results obtained demonstrates that at 10% w/w protein concentration, the error is of the order of 2% in the region of the amide I and II bands.
SusiH., in Structure and Stability of Biological Macromolecules, TimasheffS. N. and FasmanG. D., Eds. (Marcel Dekker, New York, 1969), pp. 575–663.
2.
SusiH., Meth. Enzymol.26, 445 (1972).
3.
ParkerF. S., Applications of Infrared, Raman and Resonance Raman Spectroscopy in Biochemistry (Plenum, New York, 1983).
4.
ThomasG. J.Jr. and KyogokuY., Pract. Spectrosc.1, 717 (1977).
5.
ParkerF. S. and BhaskarK. R., Appl. Spectrosc. Rev.3, 91 (1969).
6.
DeutschmannG. and UllrichV., Anal. Biochem.94, 6 (1979).
7.
ChenC. H.TowF., and BernsD. S., Biopolymers23, 887 (1984).
8.
GriffithsP. R. and de HasethJ. A., Fourier Transform Infrared Spectroscopy (Wiley, New York, 1986), pp. 338–368.
9.
KoenigJ. K. and TabbD. L., in Analytical Applications of FTIR to Molecular and Biological Systems, DurigJ. R., Ed. (Reidel, Boston, 1980), pp. 241–255.
10.
LavialleF.AdamsR. G., and LevinI. W., Biochemistry21, 2305 (1982).
11.
VincentJ. S.SteerC. J., and LevinI. W., Biochemistry23, 625 (1984).
12.
PowellJ. R.WasaczF. M., and JakobsenR. J., Appl. Spectrosc.40, 339 (1986).
13.
TherrienM.LafleurM., and PézoletM., in Proceedings of the 1985 Fourier and Computerized Infrared Spectroscopy, SPIE553, 173 (1985).
14.
PézoletM.BouléB., and BourqueD., Rev. Sci. Instrum.54, 1364 (1983).
15.
CookeR. and KuntzJ. D., Ann. Rev. Biophys. Bioeng.3, 95 (1974).
16.
PézoletM.Pigeon-GosselinM.SavoieR., and CailléJ.-P., Biochim. Biophys. Acta544, 394 (1978).
