The lumen pH was measured spectroscopically through a measurement of the electrochromic shift (ECS), which is a signal arising from the Stark effect of the electric field across the thylakoid membrane on the energy levels of carotenoids embedded in the membrane (Bailleul et al. 2010; Witt 1979). This effect causes the absorption spectrum of carotenoids in the spectral region between 450 and 550 nm to shift. The extent

of spectral shift is proportional to the amplitude of the electric field and as a result can be used to measure the transmembrane electric field. The ECS measurement can be used to probe the lumen pH by shuttering off the actinic light 4SC-202 cost and measuring the “JQ-EZ-05 reverse ECS.” Explanations of information that can be obtained from the ECS measurement, including measurements of the lumen pH, are given in Bailleul et al. (2010), Cruz et al. (2001), and Takizawa et al. (2007). To estimate the pK as of PsbS and of qZ in vivo, Takizawa and coworkers assumed that de-epoxidized xanthophyll

(i.e., zeaxanthin or antheraxanthin) and protonated PsbS are the two components necessary for qE. This assumption involved fitting to a specific mechanistic model (Fig. 4a) and excluded the possibility that the protonation of LHC proteins is a factor in qE activation https://www.selleckchem.com/products/E7080.html in vivo. Nonetheless, because it followed a specific model, this assumption enabled estimates of the pH level at which qE components were activated. The pK a of PsbS activation was fitted to be 6.8, with a Hill coefficient of ∼1, and the effective pK a of qZ was fit to be 6.8 with a Hill coefficient of 4.3. This effort is one of the first attempts thus far to fit the activation levels of Non-specific serine/threonine protein kinase qE using in vivo measurements, and the results suggest

that the pK as of PsbS and qZ are higher in vivo than the pK as for isolated glutamate (Li et al. 2002b) and for VDE in vitro (Jahns et al. 2009). Because of the challenges of estimating the lumen pH in vivo, the pK a values reported will surely be subject to refinement and reexamination. Nonetheless, the spectroscopic approach of estimating pK as and Hill coefficients is notable because the parameters are estimated from intact leaves. The approach of spectroscopically measuring the lumen pH through the ECS shift is unique and powerful in that it does not require the extraction of chloroplasts or the use of chemicals. The technique of using reverse ECS would be even more powerful it if could be extended to measure lumen pH over the course of light adaptation. Such a measurement could be used to fit mechanistic kinetic models of the protonation of the proteins involved in qE. Doing so would provide a method for determining the pK a of qE components during the process of qE induction and would enable greater precision than steady-state measurements in measuring the pK as and Hill coefficients of qE triggering.