PrOtein and ccli ETIR- ATR on silver halide fibers under reagent-accessible conditions

We described a FTIR-ATR ( Fourier transform infrared-attenuated total internal reflectance) technique for samples of 1()-3() tg deposited Ofl a p()Iycrystalline silver halide fiber ( Simhony,S.; Katzir,A .; Kosower,E.M., Anal. C/tern. 1988, 60, 1908-1910). A protein deposit consists of approximately two molecular layers, for which the kinetics and extent of exchange with D,O/N, are readily measured. For the trypsin-trypsin inhibitor complex, both rate and extent of exchange decreased with respect to those parameters fr trypsin or trypsin inhibitor individually, showing that (a) some of the NH hydrogens in the complex became inaccessible to exchange and ( b) the rate for some exchanges became slower (Chiacchiera, SM.; Kosower, EM., Anal. Biochem. 1992, 201, 43-47). We have now extended the technique in several useful ways. (a) Formation of complexes with modest association constants b' taking advantage of the combination of protein mobility on the fiber with the absence of water. Association is thus favored I ligands with proteins, e.g., D-lactose with coral tree (Ervthrina coraliodendron) lectini, and the rates and extent of deuterium-hydrogen exchange may be followed as well as conformational changes. (b) Polarized spectra of red blood cell ghosts have been measured on a flattened silver halide fiber. The cells containing the silver halide fibers have a design that allows controllable access to the sample. In addition to requiring only small quantities for good spectra, the cell is readily cleaned and handled. We anticipate the application of these techniques to many other biological samples, including mutant proteins.