base consumption in ml (or L), Nb: normality of the base,
˛: average degree of dissociation of the -NH groups, PM:mass
of protein in g (or kg), htot.: total number of peptide bonds in
meqv/g protein, and HD: hydrolysis degree %.
˛−1 is the calibration factor for the pH-stat, and it is the
reciprocal of the degree of dissociation;
˛ = 10pH−pK
1 + 10pH−pK (2)
For the calibration of the pH-stat; the amount of base consumed
during hydrolysis was recorded. The samples of
hydrolysates were taken at timed intervals and l-leucine and
l-serine equivalents were determined by TNBS and OPA reactions,
respectively. After correlation of the base consumption
with l-leucine and l-serine equivalents, slopes (b) of the
straight lines were obtained. Then, the pK values were calculated
from the following equations given by Adler-Nissen