Bioconjugation of Meldrum's acid activated furan: A detergent compatible assay for protein quantitation.

A simple yet efficient assay for the quantitation of proteins ranging from plasma proteins to purified proteins from whole cell lysate, based on the bioconjugation reaction between protein and Meldrum's acid Activated Furan (MAF) is described. This easy to use, sensitive method is based on the conjugation of amine functionalities present on the protein with MAF to form the corresponding Donor Acceptor Stenhouse Adducts (DASAs) with characteristic absorption in the visible region. The reaction is rapid as well as reproducible and shows a proportionate increase in color change over a broad range of protein concentration. The assay was found to be sensitive up to 0.125 mg/mL concentration of the protein and was compatible with most of the commonly employed detergents and isolation protocols which makes it ideal for the estimation of protein samples containing detergents. Another striking feature of this protocol is its tolerance towards other major interference contributors such as chelating agents, reducing agents, carbohydrates and protease inhibitors.

Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets.

Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 µM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria.