Hb40-61a: Novel analogues help expanding the knowledge on chemistry, properties and candidacidal action of this bovine α-hemoglobin-derived peptide.

This study expands the knowledge on chemical synthesis and properties of Hb40-61a as well as provides results of the first steps given towards knowing how it kills Candida cells. For the first time, this peptide, its all-D analogue (D-Hb40-61a) and its fluorescently labeled analogue (FAM-Hb40-61a) were successfully assembled on resin at 60°C using conventional heating in all steps. Purified and characterized, these peptides exhibited very low toxicity on human erythrocytes. Hb40-61a and D-Hb40-61a were equally active against Candida strains, ruling out sterically specific interactions on their working mechanism. Cell permeabilization assays confirmed progressive damage of the yeast plasma membrane with increasing concentrations of Hb40-61a. While experiment using the fluorescent probe DiBAC4(5) revealed that this synthetic hemocidin alters the yeast plasma membrane potential, test employing DPH indicated that Hb40-61a might affect its dynamics. Exposure of the yeast cells to FAM-Hb40-61a showed that the peptide accumulates in the cell membrane at the ½ MIC, but stains about 97% of the cells at the MIC. Such effect is salt-dependent and partially energy-dependent. These new findings indicate that the central target of Hb40-61a in Candida cells is the plasma membrane and that this synthetic hemocidin should be considered as a potential candidacidal for topic uses.

Micellar effects and analytical applications of nitro substitution in 4-Nitro-N-alkyl-1,8-naphthalimide by cysteine derivatives.

The aromatic nucleophilic substitution reactions of the nitro group of 4-Nitro-N-alkyl-1,8-naphthalimides by thiolate anions produce fluorescent derivatives and their rates are strongly accelerated by micelles of hexadecyltrimethylammonium chloride even at low pH. Acceleration factors of this reactions can reach million-fold. As the products are oxidant-insensible, this reaction allows the determination of SH- containing compounds such as cysteine, glutathione or proteins even in oxidative conditions. Limits of detection are as low as 5 × 10-7 M, ten times lower than the limit for the classic 5,5'-dithiobis-(2-nitrobenzoic) acid method. Moreover, this reaction can be developed at pHs between 6.5 and 7.5 thereby diminishing the rate of spontaneous oxidation of the thiols. In addition, we demonstrated that 4-Nitro-N-alkyl-1,8-naphthalimides can be used to evidence SH groups in peptides, proteins and living cells.