The objective of this proposal is to request funds to modify our current high performance liquid chromatography (HPLC) system so as to allow for the detection of cysteine-rich and phytochelatin heavy metal binding complexes isolated from Chlamydomonas reinhardtii. Phytochelatins (PC) are small molecular weight, cysteine-rich peptides that bind heavy metals with high affinity and specificity [Cai et al., 1999]. Recently, we have engineered Chlamydomonas cells that have an increased capacity to produce cysteine via introduction of the rice HAL2 gene into under control of the ?-tubulin promoter. These algae bind three-fold more cadmium than wild type when grown in the presence of cadmium concentrations that induce PC synthesis. This is the first engineered algae (to our knowledge) that has increased heavy metal binding capacity when grown in the presence of heavy metal concentrations that induce PC expression. As we have previously shown, heavy metal concentrations that induce PC expression increase the metal binding capacity of algae by more than 2,000 fold [Cai et al., 1999]. It is our hypothesis that algae expressing the HAL2 gene are capable of synthesizing and accumulating more PC resulting in a greater capacity to store toxic heavy metals. Alternatively, there may be other cysteine-rich amino acids or proteins (e.g. glutathione, cysteine or high molecular weight cysteine rich proteins) that bind the additional cadmium in algae expressing the HAL2 gene. The most effective method to isolate and characterize this diverse class of cysteine-rich, heavy metal binding molecules is to separate them by HPLC and to specifically detect the cysteine-rich molecules and/or phytochelatins by derivitization with 5,5'-dithio-bis(2 nitrobenzoic acid) (DTNB) [Grill et al., 1985].

The addition of the RDR-1 module to our HPLC system will allow us to efficiently detect and characterize PC and cysteine-rich molecules extracted from wild type and transgenic algae having altered heavy metal binding properties. As indicated above, we have successfully generated transgenic algae that over-express cysteine and possibly PCs. These algae have a 3-fold higher heavy metal binding capacity than wild type algae. Recently, the genes encoding phytochelatin synthetase have been isolated from plants and yeast. We now may exploit these genes for expression in algae under control of a constitutive promoter. This would allow us to induce PC expression in the absence or at low concentrations of heavy metals. We propose that algae constitutively expressing PCs may be optimal for bioremediation purposes at heavy metal concentrations that do not induce PC synthesis.

It will be critical to our understanding of heavy metal biochemistry in algae to be able to measure PC and other cysteine rich molecules that bind heavy metals. In addition, as described on the Alltec brochure, chemical derivitization methods are available to identify heavy metals separated by HPLC. Thus, we may be able to not only identify PCs and other heavy metal binding fractions but to efficiently analyze the metal content of algae using this system.

Typically, DTNB derivitization of sulhydryls (e.g., cysteine, glutathione and PCs) is done post-column or after separation of the cysteine-rich molecules by HPLC. DTNB derivitization allows one to specifically detect molecules that contain cysteine residues by measurement of their diagnostic absorbance at 410 nm or by fluorescence spectroscopy. It is noted that procedures we use for isolating cysteine-rich molecules from Chlamydomonas exclude possible contaminants (e.g. chlorophyll) that absorb light at 410 nm.

To optimally derivatize molecules separated by HPLC requires a reaction chamber that does not alter the separation profile or retention time of the separated molecules and also allows for injection of controlled quantities of the chemical modifying reagent. As a result of these constraints, the volume of reagent injected, the mixing profile, reaction time, and the temperature have to be precisely controlled to insure complete derivitization with minimal broadening of the separated peaks (cysteine containing molecules). This requires a specialized metering pump and reaction chamber system. We are requesting funds for a reaction chamber system and reagents to add to our existing HPLC system. The reaction chamber and mixing system we have in mind is an Alltec RDR-1 reagent delivery/reaction module (model number 259706, a description of the pump is attached). This is an ideal system since injection of the chemical modifying reagent is pulse-less resulting in less peak broadening.