Difference Spectral Assay for Holophytochrome

The following methodology refers only to the measurement of phytochrome in extracts and is based on the methods developed in this laboratory (31). For a discussion of in vivo phytochrome photoassay, we refer the reader to the following refer ences (19,46). Two major caveats about phytochrome measurements need to be made at this point. First, to avoid continuous photocycling, all measurements should be performed in a laboratory and kept under dim green safelight (54). Second, the validity of the phytochrome difference assay depends upon the lack of other pig

Figure 2. Absorption and difference spectra of purified recombinant oat phytochrome. (A) Absorption spectra of Pr and Pfr forms of POB and PCB adducts of recombinant oat apophytochrome A (AsphyA). (B) Difference spectra of POB and PCB adducts of recombinant AsphyA. Adapted from Murphy and Lagarias (44).

ments that absorb in the 500 to 800 nm wavelength range. For this reason, chlorophyll must be removed from extracts, from light-grown plant extracts which can be accomplished by precipitation with poly-ethyleneimine (41). The latter is not a concern with recombinant phytochromes prepared from bacterial, yeast, or insect cell extracts. Our standard method for phy-tochrome photoassay is outlined in Procedure 1. Because actinic illumination and sample measurement can be performed simultaneously, we recommend the use of a diode array spectrophotometer, such as the Model 8453 from Hewlett Packard (Wilmington, DE, USA). The length of irradiation will also depend on the light source. Typically, for a focused 250 W quartz halogen lamp, a 200-second irradiation is sufficient. The limit of sensitivity for this assay is approximately 10 nM phy-tochrome (i.e., 1 pg/mL protein).

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