PEPSIN INHIBITION BY A HIGH SPECIFIC ACTIVITY RADIOIODINATED DERIVATIVE OF PEPSTATIN

High specific activity radioiodinated derivatives of pepstatin which retain the inhibitory properties of the parent compound toward pepsin have been prepared. Pepstatin was first coupled to l-tyrosine methyl ester or l-3-iodotyrosine methyl ester. Pepstatin-l-tyrosine methyl ester was iodinated with 125I and chloramine-T and purified to homogeneity on LH20 columns. Nonradioactive pepstatin-l-iodotyrosine methyl ester and pepstatin-l-[125I]monoiodotyrosine methyl ester coeluted in gel filtration column chromatography. Using thin layer chromatography and radioautography a mixture of both these compounds also migrated as a single spot. On the basis of these criteria the radioiodinated pepstatin was judged to be virtually pure and have a specific activity of 2.16 Ci/μmol. Using the digestion of hemoglobin to assay peptic activity at pH 2.0, both pepstatin and pepstatin-l-iodotyrosine methyl ester demonstrated similar inhibition of pepsin, indicating that the addition of the iodotyrosine methyl ester group to the carboxyl terminus of pepstatin did not alter the inhibitory properties of the parent compound. By means of equilibrium dialysis at pH 5.0 in which pepstatin-l-[125I]monoiodotyrosine methyl ester was used in trace quantities, the dissociation constants KD of pepsin-pepstatin and pepsin-pepstatin-l-iodotyrosine methyl ester were determined and found to be 4.57 × 10-11 ± 1.42 m and 1.31 × 10-11 ± 0.63 m respectively. The closeness of these values to each other provides additional quantitative evidence that pepstatin-l-iodotyrosine methyl ester reacts with pepsin in a manner similar to that observed with native, unmodified pepstatin and suggests that pepstatin-l-[125I]monoiodotyrosine methyl ester may be used as an affinity label of acid proteases under the same circumstances in which native pepstatin is known to react with this class of enzymes. © 1979.