Page 119 - Algae Anatomy, Biochemistry, and Biotechnology

102 Algae: Anatomy, Biochemistry, and Biotechnology

to the protein. The proposition that these proteins could function as a near-UV-visible-light detector
dates back more than 40 years. Despite the ubiquity and ancient origins of flavoproteins, their role
in acquiring information from the radiation environment still remains a complex area of study.
Apart from difficulties in their identification, much of the reason for the lack of understanding
lies in their diversity of function. Typical absorption spectra of flavoproteins show a dominant
protein peak at 280 nm, and maior peaks at 380 and 460 nm. The overall similarity of many
blue-light action spectra with flavoprotein absorption spectra is one of the main reasons for the
belief that flavoproteins can function as blue-light photoreceptors. So far, the only biochemical
identification of flavin-based photoreceptor has been carried out in Euglena gracilis. Despite this
paucity of evidence, the hypothesis of a flavin-based photoreceptor in algae has withstood time
and still remains an accepted working hypothesis.

Action Spectroscopy

Action spectroscopy still represents the classic way to investigate photopigments. By means of
action spectroscopy the photosensitivity of a cell at different wavelengths can be measured, thus
providing information on the nature of the pigments involved in photoreception. It is still a
common belief that this approach represents the only feasible way to study the photosensory pig-
ments of a large number of species. However, direct measure of an action spectrum is much more
difficult than that of an absorption spectrum. Moreover, action spectra may not be directly corre-
lated with the absorption peaks of the pigments involved in photoreception, as light scattering
can cause several errors. When many pigments with similar absorption characteristics in the
same visible range are present, action spectroscopy often fails to discriminate between them.
Even when there is only one predominant pigment, it is not always possible to identify it. To
obtain more reliable results, threshold action spectra should be preferred to eliminate adaptation
phenomena and screening modulation, limiting the utilization of action spectroscopy to the
study of changes or increases in the photosensitivity of a mutant cell, after the exogenous addition
of a presumptive photoreceptor pigment which the cell lacks. It may be hasty to indicate the nature
of a photoreceptor only on the basis of data obtained from action spectroscopy. This is especially
true in the case of photoreceptors such as rhodopsins, which have retinal as the chromophoric
group. Retinal absorption can be fine-tuned by amino acid charges of the retinal pocket, which
allows the entire spectrum between 380 and 640 nm to be covered. Moreover, the presence or
formation of photo-intermediates may shift the absorption maxima, and make the interpretation
of the action spectrum difficult.

Absorption and Fluorescence Microspectroscopy
These techniques do not disturb the integrity of the organism or subcellular components, and allow
the examination of an uninjured system with its physiological functions intact. The spectroscopic
overshadowing of one pigment by another is avoided because each pigment is packaged in a differ-
ent structure. Thus, cellular structure can be easily correlated with pigment type by direct obser-
vation. It is possible to make exact quantitative determinations of various reactions at the time
of their occurrence in the sample, the progressive changes in these reactions, and their relationships
to different conditions in the external medium. Because of the fundamental connection between
optical parameters and properties of molecular structures, microspectroscopy allows assessments
of minute changes in the state of the molecules of various substances in the organism, the
degree of their aggregation, and the interconversions of various forms of pigments and other
important biochemical compounds with characteristic spectra. In many cases the lability and rever-
sibility of such changes make microspectroscopy the only possible method of investigation. There
is virtually no light scattering problem associated with microspectroscopic measurements, even if
the analyzed structure has a dimension of 1 mm. Obviously, the absorption spectrum cannot provide
adequate information about the photochemical action of photons as a function of their fundamental