5.7. Proteolytic Degradation

Human serum isolated from male AB plasma was used to examine the stability of the peptides to proteolysis. Peptides (30 μM) were incubated with human serum or phosphate buffered saline (PBS) at 37 °C and reactions were stopped at 0, 1, 8 or 24 h by placing the samples on ice, 6 M urea was used to denature the proteases and 20% trichloroacetic acid was used to precipitate the proteases. Peptides were separated from the proteases by centrifugation at 17,000 g for 10 min. Effects of proteolysis were examined using RP-HPLC, whereby retention times of the peptides were determined using the PBS controls at 0 h and percentage of peptide remaining in human serum was obtained from the heights of the peaks at each time point relative to 0 h. RBP-100 was the control.