Determination of trypsin inhibitor in blood plasma.

In 1922 Hussey and Northrop (1) found that trypsin and blood trypsin inhibitor formed an inactive but dissociable compound. Graphically the relationship was represented by a curve. Hussey and Northrop apparently used water as a diluent for plasma, since additions of the equivalent amounts of water, instead of the inhibitor, are indicated for the control tubes. Recently Shulman (2) used M/30 phosphate buffer in physiological saline as a diluent and found that the relationship between trypsin inhibited and the amount of the inhibitor was represented by a straight line. This meant that the blood plasma inhibitor formed with trypsin a stoichiometric, non-dissociable compound. From the work on other trypsin inhibitors (3, 4) it is known that trypsin forms non-dissociable complexes with the inhibitors within a fairly wide range of pH values from 5 to 8. The complexes dissociate below pH 3 (3, 5). The influence of hydrogen ions is therefore well established. No requirements for other ions or for the total ionic strength of the medium in which the complex is either formed or dissociated have been reported for any of the known trypsin inhibitors. In an attempt to apply the method of Kunitz (6) to the determination of trypsin inhibitor in blood plasma we have found that the salt concentrations of the diluent exerted a significant influence. A linear relationship was found only when the salt concentration of a diluent was in the range of 0.03 to 0.14 M. No specific requirements as to the nature of either cations or anions were noticed.