Figure 6.

Temporary inhibition of human cationic trypsin by wildtype SPINK1 and variant N34S. SPINK1 (25 nM) was incubated with nonsulfated (Hu1) or sulfated (Hu1-SO₄) cationic trypsin (30 nM) for 30 min, in 0.1 M Tris–HCl (pH 8.0) buffer supplemented with 1 mM CaCl₂ and 0.05% Tween 20 at 23 °C to achieve 80% decrease of trypsin activity. A, at the indicated times, 100 μl aliquots were withdrawn, and trypsin activity was measured by the addition of 2.5 μl 6 mM Suc-Ala-Ala-Pro-Lys-p-nitroanilide substrate. Trypsin activity is expressed as a percentage of the maximal activity measured in the absence of SPINK1. Data points represent mean ± SD. Symbol size may be larger than the error bars. B, at the indicated times, 200 μl aliquots were precipitated with 10% trichloroacetic acid and analyzed by 18% SDS-PAGE and Western blotting using an anti-His tag antibody, as described in Experimental procedures section. Hu1, nonsulfated cationic trypsin expressed in Escherichia coli; Hu1-SO₄, sulfated cationic trypsin purified from pancreatic juice; SPINK1, serine protease inhibitor Kazal type 1.