The use and misuse of calcium carbonate as an aid to the spectrophotometric assay of phytochrome in vitro

Supernatant and resuspended pellet samples from a centrifugation of homogenised, etiolated oat seedlings were prepared and assayed spectrophotometrically for phytochrome in the presence and absence of added calcium carbonate (CaCO₃) particles under a variety of conditions. At a constant sample thickness, in the absence of CaCO₃, increasing sample concentration had no significant effect on the expected phytochrome reading. In the presence of CaCO₃, however, as sample concentration increased, the phytochrome reading was less than, expected more so in resuspended pellet samples than in supernatant samples. At a constant sample concentration in the absence of CaCO₃, increasing sample thickness gave no significant difference from the excepted phytochrome reading in supernatant samples, but led to a slight increase over the expected phytochrome reading in resuspended pellet samples. In the presence of CaCO₃, increasing sample thickness led to a drop from the expected phytochrome reading in both sample types, but more so in resuspended pellet samples. These findings show that the use of CaCO₃ as an aid to spectrophotometric phytochrome assay can lead to large artifacts in the instrument reading and that its use should be approached with caution.