Quantification of alpha-polylysine: A comparison of four UV/Vis spectrophotometric methods

Four UV/Vis spectrophotometric methods for the quantification of α-polylysine (α-PL) are described and discussed. The methods are based on different chemical reactivities of α-PL allowing the indirect determination of α-PL concentrations in aqueous solutions down to 1–2 µg mL−1. The four methods are the trypan blue (TB) assay, the 2,4,6-trinitrobenzene sulfonate (TNBS) assay, the ortho-phthalaldehyde (OPA) assay and the bicinchoninic acid (BCA) assay. The TB assay is based on the polycationic character of α-PL in acidic aqueous solutions, in which the non-covalent binding with the oligoanionic dye TB leads to a precipitation of the dye and a concomitant decrease in the intensity of the blue color of the solution. Both the TNBS and the OPA assay utilize the nucleophilicity of the amino groups in alkaline solutions resulting in trinitrophenylated amino groups and isoindole derivatives, respectively. Finally, in the BCA assay the reductive properties of the peptide bonds are used to reduce copper(II) to copper(I) which eventually forms a stable, purple 1 : 2 complex with bicinchoninic acid. For each method, mechanistic aspects are discussed and detailed experimental protocols are provided. With respect to the level of minimum quantification the TB and the OPA assays are best.