Surprising metal binding properties of phytochelatin-like peptides prepared by protease-catalysis

Phytochelatins (PCs) consist of alternating glutamic acid and cysteine residues ([γGlu–Cys]–Xaa) and are responsible for binding to heavy metals for cellular metal homeostasis and detoxification. This paper describes the papain-catalyzed synthesis of cysteine-rich peptides as potential PC mimics. By adjusting the feed ratio of L-cysteine ethyl ester (L-Et-Cys) and L-glutamic acid diethyl ester (L-(Et)₂-Glu) followed by de-esterification, α-linked oligo(L-Glu-co-47%L-Cys) was prepared that closely matches the 1 : 1 L-Glu-to-L-Cys molar ratio of PC peptides. Plots of absorbance difference as a function of total metal-to-peptide ([M]_total–[P]_total) molar ratio were constructed for titrations with Zn(II), Cd(II), Co(II) and Ni(II). A series of equations were generated to evaluate dissociation constants and the number of metal ions per peptide molecule in metal–peptide complexes. All of the four complexes have on average two oligo(L-Glu-co-47%L-Cys) molecules per divalent cation. The binding of metals to oligo(L-Glu-co-47%L-Cys) weakens in the order of Cd(II) > Zn(II) > Ni(II) > Co(II), the same trend observed for [(γGlu–Cys)₄]–Gly. The peptide quantity required to sequester a given amount of Zn(II) and Cd(II) when using oligo(L-Glu-co-47%L-Cys) is only about twice as much as the quantity for perfect sequence peptide (γGlu–Cys)₂–Gly. Furthermore, when [M]_total is low, random oligo(L-Glu-co-47%L-Cys) sequesters much higher fractions of Co(II) and Ni(II) than does (γGlu–Cys)₂–Gly. Results from this work provide the first evidence that, in some cases, uniform peptides synthesized by tedious solid or liquid phase peptide synthetic methods, can be replaced with peptide mixtures prepared by facile protease-catalyzed peptide synthetic methods without substantial loss in product performance.