The development of a stopped-flow instrument that operates over a temperature range of -40 to +100 °C and up to 200 MPa is described. The system has been designed so that measurements can be performed in absorbance and fluorescence modes simultaneously, without dismantling the unit. It can easily be combined with an optical system of a conventional ambient pressure setup by using light guides. Optimum optical performance and a wide operating wavelength range (220-850 nm) are achieved as the light is not passing through the pressurizing fluid. A special design for the pistons has been developed; thus, the apparatus has proven to be leak-free, even under extreme conditions (high pressure, low temperature, various solvents). The dead time of the system is found to be less than 2 ms at 298 K and is pressure independent up to 200 MPa. We examined the kinetics for the formation of the Mg(2+)-8-hydroxyquinoline chelate in aqueous solutions at pH 8.0 in order to develop a convenient alternative test method for high-pressure stopped-flow spectrometers with absorption and fluorescence detection.

Stopped-flow kinetic studies have been performed to determine the kinetic parameters of K+ binding to the fluorescent cryptand F222 and of Na+binding to F221 at pH 8.O. The results clearly indicate that a comparatively stable intermediate is formed before the rate-limiting binding step occurs with a rate constant around 30 s−1 under the chosen experimental conditions. The conversion of the intermediate to the final cation complex is assigned to the final penetration of the already bound, but still partially solvated cation into the ligand's cavity. The main fluorescence intensity change found upon cation binding is attributed to the second reaction step, and not to the fast, initial binding reaction. The comparatively slow overall binding reaction is interpreted on the bases of a special solvate substitution mechanism which, in principle, can also account for the 1500 times slower binding of Ca 2+ to F221. With regard to time-resolved analytical Na+ and K+ determinations, the response times under the chosen conditions are around 20 ms. Differentiation between Na+ and Ca2+, for example, is possible with F221 on the basis of completely different response times.

Most mechanistic aspects of cation binding and transport by Na+K+-ATPase are still unknown. The kinetics of cation binding with the FTTC-enzyme can be studied using the stopped flow technique (1,3,4) taking advantage of the large fluorescence intensity changes caused by these reactions (1,2). In contrast to equilibrium titration experiments, kinetic studies can provide evidence for the existence of intermediate states.

To link proposed features of the Na+/K+-ATPase reaction cycle to the molecular structure, spectroscopic studies mainly concerning the elucidation of partial reactions are carried out. This can be done by introducing fluorescence labels such as the F1TC-group (1) which enables the study of alkali ion binding as a key feature of this ion pump. According to earlier studies, a correlation between the fluorescence emission intensity and the position of the E1/E2 equilibrium is suggested: low fluorescence intensity in the presence of K+ has been attributed to E2, high intensity to E1(1–5). Evidence will be presented that this correlation concerning E1 is not fulfilled. Membrane-bound Na+/K+-ATPase has been prepared from pig kidney according to (6), the FITC-enzyme according to (1); details are given in (7).