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.