Gel-filtration chromatography using β-tryptase supernatant. A total of 400 mL of individual samples (α- and β-tryptase intracellular or secreted forms) were loaded on a Superdex 200 HR preparative grade gel filtration column, and 1-mL fractions were collected and analyzed for tryptase enzymatic activity as described in the Materials and Methods. The elution profile of three reference protein standards (BSA [67 kD], aldolase [158 kD], and catalase [232 kD]) is depicted. A single, maximal peak of hydrolytic activity comigrating with the known homotetrameric form of β-tryptase (∼130 kD) is evident, with no hydrolytic activity seen at other molecular weights. Identical (but smaller) peaks of proteolytic activity were seen using both secreted and intracellular forms of α-tryptase but not intracellular β-tryptase (not shown).