Page 120 - Algae Anatomy, Biochemistry, and Biotechnology

Anatomy 103

energy; however, the identification of the chromophores in the photoreceptive structures does
provide information about possible mechanisms of energy transfer. The measurements are very dif-
ficult when small changes in absorption have to be measured in the presence of a strong total signal
(luminous background), as photon noise is proportional to the square root of the intensity of the
incident light. Then, fluorescence spectroscopy is recommended: it can achieve more reliable
results compared with absorption spectroscopy, because the background emission is much
reduced. In this case the sensitivity of detection is not limited by the signal-to-noise ratio, but
rather by the presence, virtually unavoidable, of fluorescent contaminants.

Biochemical and Spectroscopic Study of Extracted Visual Pigments
Extraction of visual pigments (chromophore or protein), either by means of detergents such as digi-
tonin and TRITON or by organic solvents, could be the best method for providing large quantities
of photoreceptive pigments in an accessible in vitro form for subsequent detailed biochemical
analysis. Such samples allow a very accurate determination of spectroscopic parameters. Spec-
troscopy of solubilized pigment may be complicated, however, by the simultaneous extraction of
several other pigments in the cell, which cause distortion of absolute spectra and necessitate
special procedures of purification. These problems can be solved by separating the different pig-
ments after extraction, for example, by high performance liquid chromatography (HPLC) and
final identification with gas chromatography – mass spectrography (GC–MS) for chromophores
or affinity chromatography for proteins. As pigment extraction permanently removes the identify-
ing link to a particular cell structure and may change the spectroscopic properties of a receptor
because native interactions are disrupted, these detrimental factors mandate a careful evaluation
of the results obtained by this method.

Electrophysiology

Light excitation of the photoreceptor generates a cascade of electrical events. Electrophysiology
was the elective method in the study of vertebrate photoreceptors. However, this technique has
been applied with less success in the algae because it is very difficult to locate the photoreceptors
in the cell body and, when this is possible, to produce a good sealing between the glass pipette and
the cell membrane.
Flash-induced transient depolarizing potentials using intracellular glass microelectrode were
first identified in Acetabularia crenulata. However, the first detailed analysis of photocurrents
were possible by employing a suction pipette technique (patch clamp technique) in Haematococcus
pluvialis and in the wall-free mutants of the unicellular green alga Chlamydomonas sp. In these
experiments whole cells were gently sucked into fire-polished pipettes, forming seals with resist-
ances up to 250 MW, allowing cell attached recordings from a relatively large membrane area,
though higher resistance seals were not achieved. Recently, Negel et al. (2002) demonstrated by
means of electrophysiology that the rhodopsin-like protein of Chlamydomonas, expressed in
Xenopus laevis oocytes in the presence of all-trans retinal produces a light gated conductance
that shows characteristics of a channel selectively permeable for protons.

Molecular Biology Investigations
DNA hybridization is useful in attempting to determine phylogenetic interrelationship between
species. The rationale is that similarities between DNA structures correlate to interrelatedness.
It is used to detect and isolate specific sequences and to measure the extent of homology between
nucleic acids. It represents an alternative to the study of visual pigments at the protein level, as
the genes encoding these proteins can be identified, their sequences determined, and the comparative
genetic information assessed. Genomic Southern blot hybridization is used to probe the genomes of
a variety of species in a manner analogous to that reported for other protein families. The potential