TY - JOUR
T1 - Deuterium exchange on micrograms of proteins by attenuated total reflection fourier transform infrared spectroscopy on silver halide fiber
AU - Chiacchiera, Stella M.
AU - Kosower, Edward M.
N1 - Funding Information:
The authors are grateful to the Israel-United States Binational Research Foundation and the Basic Research Foundation of the Israel Academy of Sciences for support. Partial fellowship support was provided by Consejo National de Investigaciones Cientificas y Tecnicas (CONICET), Argentina. Special thanks are due to Professor Aharon Katzir and his group in the School of Physics, Tel-Aviv University, for much help and cooperation as well as for the supply of the silver halide fibers.
PY - 1992/2/14
Y1 - 1992/2/14
N2 - We illustrate the use of polycrystalline silver halide fibers (2-20 μm transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 μg 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) - Σ [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.
AB - We illustrate the use of polycrystalline silver halide fibers (2-20 μm transparency range) for attenuated total internal reflection Fourier transform infrared (IR) spectroscopic measurements of microsamples (10 μg 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) - Σ [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.
UR - http://www.scopus.com/inward/record.url?scp=0026575409&partnerID=8YFLogxK
U2 - 10.1016/0003-2697(92)90171-3
DO - 10.1016/0003-2697(92)90171-3
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AN - SCOPUS:0026575409
SN - 0003-2697
VL - 201
SP - 43
EP - 47
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -