A dialysis accessory for attenuated total reflection (ATR) infrared spectroscopy is described together with an evaluation based on known systems with well-studied infrared spectra, such as chemical oxidation and reduction of cytochrome c and substrate binding to the Ca^2+-ATPase. Changes in the infrared spectra of the two proteins are successfully monitored with the dialysis accessory. The accessory was developed in our laboratory for the diamond 9-reflections SensIR ATR unit. It can be used to study absorbance changes of macromolecules which are induced by low molecular weight compounds, for example the binding of substrates, inhibitors or ions to macromolecules as well as effects of pH, ionic strength or denaturants on the structure of macromolecules. The dialysis accessory confines the macromolecule of interest to a sample compartment created between the ATR crystal and the dialysis membrane. On the other side of the dialysis membrane, a reservoir for the sample medium is created. In this way the low molecular weight compound of interest can exchange freely between the reservoir and the sample compartment via the dialysis membrane. This provides a flexible way to change sample conditions for the macromolecule of interest, allowing for example initiation of ligand binding.

Structural changes in rabbit muscle pyruvate kinase (PK) induced by phosphoenolpyruvate (PEP) and Mg2+ binding were studied by attenuated total reflection Fourier transform infrared spectroscopy in combination with a dialysis accessory. The experiments indicated a largely preserved secondary structure upon PEP and Mg2+ binding but also revealed small backbone conformational changes of PK involving all types of secondary structure. To assess the effect of the protein environment on the bound PEP, we assigned and evaluated the infrared absorption bands of bound PEP. These were identified using 2,3-C-13(2)-labeled PEP. We obtained the following assignments: 1589 cm(-1) (antisymmetric carboxylate stretching vibration); 1415 cm(-1) (symmetric carboxylate stretching vibration); 1214 cm(-1) (C-O stretching vibration); 1124 and 1110 cm(-1) (asymmetric PO32- stretching vibrations); and 967 cm(-1) (symmetric PO32- stretching vibration). The corresponding band positions in solution are 1567, 1407, 1229, 1107, and 974 cm-1. The differences for bound and free PEP indicate specific interactions between ligand and protein. Quantification of the interactions with the phosphate group indicated that the enzyme environment has little influence on the P-O bond strengths, and that the bridging P-O bond, which is broken in the catalytic reaction, is weakened by <3%. Thus, there is only little distortion toward a dissociative transition state of the phosphate transfer reaction when PEP binds to PK. Therefore, our results are in line with an associative transition state. Carboxylate absorption bands indicated a maximal shortening of the length of the shorter C-O bond by 1.3 pm. PEP bound to PK in the presence of the monovalent ion Na+ exhibited the same band positions as in the presence of K+, indicating very similar interaction strengths between ligand and protein in both cases.

Pyruvate kinase exhibits allosteric properties. The allosteric effect of fructose 1,6-bisphosphate (FBP) on phosphoenolpyruvate (PEP) binding to rabbit muscle pyruvate kinase (PK) in the presence of various ions (mg(2+), mn(2+), Na(+)) was studied by attenuated total reflection infrared spectroscopy in combination with a dialysis accessory. The experiments indicated that FBP binding causes conformational changes of PK that are of the same order of magnitude as those of PEP binding. The conformational change of PEP binding to PK/Mg(2+)/K(+) in the presence of FBP was about twice as large as in its absence, which is tentatively ascribed to a higher occupancy the closed state. The affinity for PEP increased in the presence of Mg(2+) and K(+). No such effects were observed with the other ion combinations Mn(2+)/K(+) and Mg(2+)/Na(+) or in D(2)O (with Mg(2+)/K(+)), and therefore we did not detect an allosteric effect on PEP binding under these conditions.