Monitoring tripeptidase activity using capillary electrophoresis. Comparison with the ninhydrin assay.

Capillary electrophoresis (CE) was used to assay the activity of a tripeptidase from a crude extract of Lactococcus lactis subsp. lactis NCDO 712 against the substrate, Gly-Gly-Phe and a comparison with a standard ninhydrin assay was made. Standard curves of the substrates and products showed a significantly variable colorimetric reaction to ninhydrin making accurate quantification of the tripeptidase problematic. The CE assay further demonstrated that the presence of contaminating enzymes in crude cell-free extracts can cause secondary reactions that are not apparent from the ninhydrin assay data. The CE assay was also able to generate enzyme kinetics data and monitor, during purification, the presence of co-eluting contaminating activities. The speed and sensitivity with CE allows routine analysis of the tripeptidase activity without any derivatization normally required for this enzyme.

pH- and ionic strength-dependent fusion of phospholipid vesicles induced by pardaxin analogues or by mixtures of charge-reversed peptides.

The fusogenic properties of the neurotoxin paradaxin and eight of its analogues with small unilamellar vesicles (SUV), composed of egg phosphatidylcholine and phosphatidylserine (PC/PS), were investigated. Fusion was demonstrated by a lipid-mixing assay and by an increase in vesicle size as revealed by electron microscopy. The lipid-mixing assay was performed at either neutral (pH 6.8) or acidic (pH 4.5) conditions, in solutions containing either high or low salt concentrations. A low level of fusion could be induced at neutral pH only by pardaxin derivatives with amino groups at both the peptide's backbone and N-terminus. However, a marked enhancement in the fusogenic activity occurred when amino groups were present also in the C-terminus. Pardaxin analogues in which amino groups were substituted by carboxylic groups induced elevated levels of fusion only at high salt concentrations where enhancement of aggregation occurs, and acidic pH, which increased alpha-helicity. The influence of mutual interactions between pardaxin's analogues possessing complementary charges on the lipid-mixing process was also studied. At neutral pH and high salt, an inactive acidic analogue increased the fusogenic activity of a complementary-charged basic peptide. However, such mutual interactions at low salt concentrations reduced the fusogenic activity of the pardaxin analogues. Analogues containing D-amino acids were not fusogenic, thus demonstrating the structural specificity of these observations. The results indicate that the charge, alpha-helical structure, and aggregation of peptide monomers play an important role in the fusogenic ability of polypeptides.