The late intermediates involved in the activation mechanism of bovine rhodopsin are investigated by time-resolved optical absorption spectroscopy. Measurements from 10 μs to 200 ms after photolysis were carried out on membrane suspensions of bovine rhodopsin at a temperature of 15°C and at pH of 7.3, 8.0, and 8.7. The time-resolved absorption spectra in the 330−650 nm range were analyzed by global exponential and kinetic scheme fitting methods. The results indicate an activation mechanism that

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... s more complex than suggested previously. It involves interconnected branched pathways with two metarhodopsin I 480 and two metarhodopsin II intermediates. The intermediates involved in this more complex mechanism need to be considered in spectroscopic studies that vary sample temperature and pH in order to enhance the presence of specific rhodopsin intermediates.