ABSTRACT
Phytochelatins (PCs, (gamma Glu-Cys)(n)-Gly, n=2-11) are produced by higher plants, algae and some fungi in order to detoxify Cd(2+) by sequestration to form Cd-PCs complexes. In order to investigate what chemical structures of PCs are responsible for their metal-binding ability, various cysteine-rich peptides ((X-Cys)(7)-Gly, X=Glu, Asp, Lys, Gly, Ser and Gln) were chemically synthesized. Water-solubility, metal-binding property, and detoxification effect toward Cd(2+) were analyzed and compared with those of (gamma EC)(7)G. (SC)(7)G and (QC)(7)G were insoluble at pH below 10, and (GC)(7)G was not soluble at any pH between 1 and 12, indicating that charged side chains were at least required for the molecules to be solubilized in aqueous solution. By spectroscopic analyses using DTNB method and UV method, we found that (EC)(7)G and (DC)(7)G had almost equivalent abilities of Cd(2+)-binding as PC ((gamma EC)(7)G), indicating that the distance between each thiol group was not a major factor for the binding to Cd(2+). (beta DC)(7)G and (KC)(7)G interacted to Cd(2+) with fourth coordination as in the case of other soluble PC-related peptides. However, compared to (gamma EC)(7)G, (beta DC)(7)G displayed a slightly weaker binding to Cd(2+), and (KC)(7)G showed a drastic decrease in binding ability. The affinities of PC-related peptides toward Cd(2+) were evaluated as below; (gamma EC)(7)G=(EC)(7)G=(DC)(7)G>(beta DC)(7)G>>(KC)(7)G=weak binding. The results of Cd(2+)-detoxification assays were consistent with the affinity between Cd(2+) and the peptides. We concluded that the structure consisting of thiol and carboxyl groups were essential for the formation of a tight Cd-peptides complex such as Cd-PCs.
Subject(s)
Chelating Agents/metabolism , Chelating Agents/pharmacology , Metalloproteins/metabolism , Metalloproteins/pharmacology , Metals/metabolism , Peptides/metabolism , Peptides/pharmacology , Plant Proteins/metabolism , Plant Proteins/pharmacology , Cadmium/metabolism , Chelating Agents/chemistry , Environmental Pollutants/metabolism , Glutathione , In Vitro Techniques , Inactivation, Metabolic , Metalloproteins/chemistry , Peptides/chemical synthesis , Peptides/chemistry , Phytochelatins , Plant Proteins/chemistry , Protein Binding , SolubilityABSTRACT
Phytochelatins (PCs, (gammaGlu-Cys)n-Gly (n = 2-11)) are produced by higher plants, algae, and some fungi in response to heavy metal ion exposure. A rapid and convenient method for quantifying heavy metal ion concentrations in water environments was developed using a chemically synthesized PC as a mediator. The chelating ability of the PC and quantification of the thiol group were utilized to measure heavy metal ions at low concentrations. The method requires only ten minutes for measurement and only 1 ml of a liquid sample. A range of homogeneous PCs (n = 4-7) were chemically synthesized using a peptide synthesizer. These, especially PC7, exhibited higher sensitivity and consistency of measurement than the native PC from Silene cucubalus, which produced a mixture of PC2, PC3, and PC4. Detoxification of heavy metal ions in vitro by PC was also investigated. Using the paper disc method, the cell growth inhibition zone caused by cadmium ion against Salmonella typhimurium TA1538 was significantly decreased by addition of PC. Furthermore, at the minimum inhibitory concentration of cadmium ion (200 microM) in a nutrient broth culture of S. typhimurium, cell growth was almost completely recovered by addition of PC to the medium.
