Materials and Methods

Protein Purification. CT inhibitor was isolated from human plasma by precipitation with polyethylene glycol), chromatography on DEAE-cellulose and hexyl-Sepharose (Nilsson & Wiman, 1982), and gel filtration in 0.1 M NH4HC03, pH

8.3, on Sepharose 6B. The resulting single-chain material was fully active against Cls (Chapuis et al., 1977) and migrated with an apparent molecular mass of 105 000 Da in NaDod-S04-PAGE. Phosphate was determined by a modified Fiske-Subbarow method (Ames, 1966). The activation pep-tide was separated from the Cls-CT inhibitor complex by gel filtration on Sephacryl S-300 in 40 mM sodium phosphate, 0.1 M NaCl, 0.1 M NaN3, and 0.1% NaDodS04, pH 7.3. Amino Acid Sequencing. CT inhibitor was degraded chemically or enzymatically as indicated in Figure la. For digestion with pepsin, the protein was dissolved in 99% formic acid and then diluted to 5%; the enzyme/substrate ratio was

1/100 w/w, and the digest was incubated at room temperature for 3 h. Carbohydrate-rich peptides were treated with tri-fluoromethanesulfonic acid (Edge et al., 1981) or alkaline sodium borohydride (Spiro & Bhoyroe, 1974). Peptides were purified by an initial gel filtration on Sephadex G-50F or Sephacryl S-200, mostly in 0.1 M NH4HC03, followedby ion-exchange chromatography on DEAE-Sephacel using a linear gradient of 0.01-1.0 M NH4HC03. Final purification was achieved by reversed-phase HPLC on a Hewlett-Packard 1084B liquid chromatograph. Peptides were sequenced on an

Applied Biosystems Model 470A (using the chemicals and the 02n vac program supplied by the manufacturer) or on a Beckman 890C sequenator. Amino acid analysis was per-formed on a Beckman 121MB instrument. In addition to the systems commonly used in our laboratory (Skorstengaard et al., 1982, 1984), columns of Vydac C4 (with elution gradients of 2-propanol and triethylamine, pH 5.2, in 0.1% CF3COOH)