The net charge values of the peptides at pH 4.8, 7.4, and 10.5 , where Z is the net charge, Ni is the number of positively charged groups in the peptide, i.e. the histidines and the N-terminus, depending on the peptide, Nj is the number of negatively charged groups, i.e. the C-terminus, and pKa,i or pKa,j are their corresponding pKa values. The absolute net charge was calculated by adding the two components in the equation rather than subtracting them.