Deuterium exchange on micrograms of proteins by attenuated total reflection Fourier transform infrared spectroscopy...

We illustrate the use of polycrystalline silver halide fibers (2-20 microns transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 micrograms of protein). A powerful adjunct technique is a simple method for carrying out deuterium for proton exchange. Spectra of trypsin, soybean trypsin inhibitor, and their complex are easily obtained. Two kinds of difference spectra (DS) are revealing: DS1 (changes in protein on combination with ligand), IR of the trypsin-soybean trypsin inhibitor complex (T.SBTI complex)--sigma [IR of trypsin (T) + IR of soybean trypsin inhibitor (SBTI)], the small values at all wavelengths indicating no conformational change of the proteins upon complexation, and DS2 (changes in materials on deuteration), IR of protioprotein--IR of deuterioprotein, which reveals the infrared bands affected by deuteration. The rate and the extent of the exchange are additional valuable parameters readily measured with this technique. In the present instance, the rate and the amount of the exchange for T.SBTI complex after 30 min was substantially less than that expected from the simple sum of the same parameters for the two individual proteins, T and SBTI. The enzymatic activity of trypsin on the fiber survived for more than a day, no autodegradation being detected by SDS-gel electrophoresis.