A RAPID SPECTROPHOTOMETRIC METHOD FOR MEASURING PHOTOSYSTEM-I AND PHOTOSYSTEM-II ACTIVITIES IN A SINGLE-SAMPLE

A spectrophotometric method has been developed for the sequential meas urement of photosystem II (PSII) and photosystem I (PSI) activities in a single sample of thylakoid membranes. The assay is based on the red uction and subsequent oxidation of DCPIP (2,6-dichlorophenolindophenol ) by PSII and PSI respectively. Electrons donated from water reduce DC PIP during the PSII reaction, whereas reduced DCPIP acts as the electr on donor and methyl viologen as the electron acceptor during the assay of PSI. DCPIP absorbs strongly at 580 nm in oxidized form, but it los es absorbance when it is reduced, thus providing the potential for a s pectrophotometric assay of both photosystems. Although PSII activity i s commonly monitored spectroscopically using DCPIP, PSI is traditional ly assayed polarographically as O-2 consumption. However, PSI activiti es obtained with the spectrophotometric method we describe, using thyl akoid samples equivalent to only 10 mu g of chlorophyll, were signific antly higher than measurements obtained with 30-mu g samples using a s tandard oxygen electrode. Ascorbate was added to the PSI reaction mixt ure to complete the reduction of DCPIP before PSI measurement. and DCM U was included to block electron transport from PSII to PSI. The highe st activities were recorded at pH 8 for PSI and at pH 7 for PSII. Neve rtheless, values that approximated or exceeded 80% of the peak activit ies of both photosystems were obtained at pH 7.5, which allowed sensit ive, sequential measurement of PSII and PSI in a single cuvette.