Application notes 15 min read

Measurement of electrochromic bandshift at 520 nm with real-time scatter correction at 546 nm

Latest updated: January 9, 2020

Introduction
The formation of a transmembrane electric field during photosynthesis leads to absorbance peak shifts of certain pigments in the antenna of photosynthetic organisms. These field-indicating absorbances were first reported in 1968 by Junge and Witt [1], and are a direct observation of the trans-thylakoid voltage so their rates indicate current, i.e. they are simultaneously a molecular voltmeter and an ammeter. Since the field indicating absorbance is only dependent on the formation of the field, the response is immediate, and the voltage and current are indicated linearly and can be calibrated to yield absolute values. The field-indicating absorbance changes are induced via electrochromism. For a pigment in solution, electrochromism has three effects, (1) changes in angle between the transition dipole and the field orientation, or the orientation effect, (2) direct effects of the field on the transition dipole moment, or transition moment effect,and (3) shift in absorption band, or bandshift effect.

 

For protein-bound pigment in a membrane, the bandshift effect describes the behavior, and a simplified model is seen in figure 1. In panel A, a pigment is excited by a photon and is raised to an excited energetic state. In the absence of an external voltage this will occur with a wavelength equal in energy to the electronic transition, however if either of the ground or excited states have differences in dipole moment or polarizability, then an external field will change the energy of the transition. The corresponding excitation wavelength will likewise change. Figure 1B demonstrates the effect on the peak position for a situation in which the pigment has been electrochromically shifted to a lower transition energy, i.e. red shifted. In the case of a sample in which the baseline was collected in the absence of an electric field, and then measured in the presence of the field, the result is the green trace in 1B. In photosynthetic organisms this field is rapidly established in the light and diminished in the dark and can therefore be used as an inherent probe of many membrane voltage dependent phenomena. This “bandshift effect” is the cause of the “field-indicating absorption changes, and is commonly referred to as the electrochromic shift (ECS) in photosynthesis research.