Mechanism of Na(+) binding to thrombin resolved by ultra-rapid kinetics

Abstract

The interaction of Na(+) and K(+) with proteins is at the basis of numerous processes of biological importance. However, measurement of the kinetic components of the interaction has eluded experimentalists for decades because the rate constants are too fast to resolve with conventional stopped-flow methods. Using a continuous-flow apparatus with a dead time of 50 micro s we have been able to resolve the kinetic rate constants and entire mechanism of Na(+) binding to thrombin, an interaction that is at the basis of the procoagulant and prothrombotic roles of the enzyme in the blood.

SCHEME 1

Figure 1

A,B (A) Kinetic traces of Na⁺ binding to human thrombin in the 0–250 ms time scale. Shown are the traces obtained at 50 and 100 mM Na⁺ with the stopped-flow method using an Applied Photophysics SX20 spectrometer, with an excitation of 280 nm and a cutoff filter at 305 nm [8]. Traces are averages of three determinations. Binding of Na⁺ obeys a two-step mechanism, with a fast phase completed within the dead time (<0.5 ms) of the spectrometer, followed by a single-exponential slow phase. The k_obs for the slow phase decreases with increasing [Na⁺] (Figure 2A). (B) Kinetic traces of Na⁺ binding to human thrombin in the 0–700 μs time scale. Shown are the traces obtained at 36 and 91 mM Na⁺ with the continuous-flow method as single determinations with an exposure time of 3 s. We built an ultra-rapid mixer [23] of similar design and methodology to that described by Shastry and Roder[24, 25]. The flow cell was purchased from Hellma (Germany). The mixed sample was illuminated with an A1010B Mercury-Xenon lamp (PTI, UK) at 280 nm, using a Model 101 6 Monochromator (PTI, UK). Fluorescence was recorded with a Micromax CCD camera (Princeton Instrument, USA), with a typical exposure time of 1–3 s and employing different emission glass filters. An original, pneumatically driven, loading syringes unit was designed. Data were recorded at 3 bars, leading to a linear velocity in the flow cell of about 16 m/s. Experimental conditions were identical to those used in the stopped-flow experiments. The dead time of the instrument was determined by fluorescence using the quenching of N-acetyl-tryptophanamide (NATA) by N-bromo-succinamide (NBS) [26]. The pseudo-first-order quenching reaction of NATA by NBS was measured at NBS concentrations from 3.5 to 33 mM. The observed fluorescence traces obtained at different NBS concentrations yielded single exponential time courses extrapolating to a common point near fl_rel = 1, the expected initial fluorescence of NATA in the absence of quencher. The time delay from this point to the first data point that falls on the fitted exponential provided an estimate of the dead time, 40–50 μs, of the instrument. Binding of Na⁺ in the 0–700 μs time scale obeys a single-exponential phase with a k_obs increasing linearly with [Na⁺] (Figure 2B). This resolves the fast phase detected with the stopped-flow method and shown in (A). Experimental conditions for the two methods are: 5 mM Tris, 0.1% PEG8000, pH 8.0 at 25 °C. The thrombin concentration was 50 nM for the stopped-flow measurements and 40 μM for the continuous-flow measurements. The [Na⁺], as indicated, was changed by keeping the ionic strength constant at 400 mM with choline chloride. Continuous lines were drawn using the expression a + bexp(−k_obst) with best-fit parameter values: (A) a=8.944±0.001 V, b=−0.10±0.01 V, k_obs=111±9 s⁻¹ ([Na⁺]=50 mM); a=9.427±0.001 V, b=−0.29±0.01 V, k_obs=96±6 s⁻¹ ([Na⁺]=100 mM). (B) a=0.417±0.002, b=−0.072±0.02, k_obs=7±2 ms⁻¹ ([Na⁺]=36 mM); a=0.477±0.002, b=−0.10±0.03, k_obs=10±2 ms⁻¹ ([Na⁺]=91 mM). All data were collected at least in duplicate.

Figure 2

A,B Values of k_obs vs [Na⁺] for the slow and fast phases of fluorescence change due to Na⁺ binding to thrombin shown in Figure 1. Shown are the results pertaining to the stopped-flow (A) and continuous-flow (B) measurements. Note the different time scale between the two panels. Experimental conditions are given in the legend to Figure 1. Continuous lines were drawn according to eqs 1 and 2 in the text, with best-fit parameter values: k₁=67±7 s⁻¹, k₋₁=69±6 s⁻¹, k_A=56,000±200 M⁻¹s⁻¹, k_−A=4,800±200 s⁻¹.