Extraction of the Functional Manganese and Calcium From Photosystem II

Manganese (Mn) and calcium (Ca) are both metal cofactors required for photosynthetic oxygen evolution (1 ). The functional roles for these ions in the O₂ -evolving reactions are not completely known. They are studied by comparative spectroscopic measurements between intact and metal-depleted samples. In this chapter, we outline three experimental procedures used for the various removal of Mn and Ca from photosystem (PS) II-containing (i.e,. O₂ -evolving) preparations: the complete Mn extraction using a strong alkaline Ches buffer (pH 9.4)/MgCl₂ wash, partial Mn extraction using a mild hydroxylamine (pH 6.8) wash, and specific Ca extraction through a low pH/citrate (pH 3) wash. The O₂ evolution activities (measured by a Clarke-type oxygen electrode), protein composition (determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis), and the relative Mn and Ca content (measured by atomic absorption spectroscopy) are reported for each extraction procedure.