pH gradients across thylakoid membranes measured with a spin‐labeled amine

In their uncharged form amines have been shown to be permeable across membranes and to establish concentration gradients proportional to H’ gradients [ 1,2] . In their protonated form they may bind to negative moieties on the surface of proteins or lipid layers. Amines have therefore been used to study pH gradients across membranes [2-91 as well as the surface charge of membranes [ 1 O-l 31 . In particular the quenching of 9-aminoacridine fluorescence has been used to measure the pH gradient across thylakoid membranes [4,8], while that changes in the fluorescence of acridines are claimed [ 10,12,13] to be closely associated with their strong interaction with the membrane surface and that transport across the thylakoid membrane may not be required. When using amine probes to measure pH gradients across membranes it becomes important to distinguish between the free and membrane bound populations of the probe; we have therefore used the spin-labeled amine 4-amino-2,2,6,6-tetramethyl-piperidine-N-oxyl (Tempamine) with pK = 9.5, which shows distinct bound and free EPR signals. An impermeable analogue,4-trimethylammonium-2,2,6,6-tetramethylpiperidine-N-oxyl bromide (CATi) and the impermeable spin broadening agent K,Fe(CN), were used to demonstrate that it is the uncharged form of Tempamine that is permeable to the thylakoid membrane. The permeable uncharged spin label 2,2,6,6-tetramethyl+piperidone( “N-Tempone) was used to show that at low concentrations of Tempamine (150 PM) virtually no swelling of the thylakoids occurred during illumination. TEMPAMINE CAT, 15N TEMPONE