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Biochem. J. (1984) 221, 445-452 445 Printed in Great Britain Human plasma a-cysteine proteinase inhibitor Purification by affinity chromatography, characterization and isolation of an active fragment Anne D. GOUNARIS,* Molly A. BROWN and Alan J. BARRETT Department ofBiochemistry, Strangeways Laboratory, Worts Causeway, Cambridge CBJ 4RN, U.K. (Received 16 January 1984/Accepted 5 April 1984) Human plasma a-cysteine proteinase inhibitor (aCPI) was purified by a two-stage method: affinity chromatography on S-carboxymethyl-papain-Sepharose, and high- resolution anion-exchange chromatography. The protein was obtained as a form of Mr about 64000 and material of higher Mr (about 100000). In sodium dodecyl sulphate/polyacrylamide-gel electrophoresis with reduction, both forms showed a major component of M, 64000. An antiserum was raised against aCPI, and 'rocket' immunoassays showed the mean concentration in sera from 19 individuals to be 35.9mg/dl. Both low-Mr and high-Mr forms of aCPI were confirmed to be sialoglyco- proteins by the decrease in electrophoretic mobility after treatment with neuramini- dase. aCPI was shown immunologically to be distinct from antithrombin III and a,-antichymotrypsin, two serine proteinase inhibitors from plasma with somewhat similar Mr values. aCPI was also distinct from cystatins A and B, the two intracellular low-Mr cysteine proteinase inhibitors from human liver. Complexes of aCPI with papain were detectable in immunoelectrophoresis, but dissociated to free enzyme and intact inhibitor in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The stoichiometry of binding of papain was close to 1:1 for both low-Mr and high-Mr forms. aCPI was found to be a tight-binding inhibitor of papain and human cathepsins H and L (Ki 34pM, 1.1 nM and 62pM respectively). By contrast, inhibition of cathepsin B was much weaker, Ki being about 35 uM. Dipeptidyl peptidase I also was weakly inhibited. Digestion of aCPI with bromelain gave rise to an inhibitory fragment of Mr about 22000, which was isolated. The best known ofthe cysteine proteinases (once known cysteine proteinases fall into the super- called 'thiol' proteinases) is papain, and most ofthe family of proteins homologous with papain (Bar- rett et al., 1984). Since this group contains the major lysosomal proteinases cathepsins B, H and Abbreviations used: the abbreviations used for names L, as well as chymopapain, which is used medic- of amino acid derivatives and N-terminal groups are based on the standard conventions [Biochem. J. (1984) ally, the physiological inhibitors ofthe enzymes are 219, 345-373]. The C-terminal groups are: CH2CL, of interest. Of the small number of proteins known chloromethane, NPhNO2, p-nitroanilide, and NMec, to act as tight-binding inhibitors of these cysteine 7-(4-methyl)coumarylamide. Other abbreviations used proteinases, all are small proteins, Mr about 13000, are: Cm-, S-carboxymethyl; aCPI, plasma a-cysteine except the plasma protein, aCPI. proteinase inhibitor; E-64, L-3-carboxy-2,3-epoxypro- Human aCPI was first described by Sasaki et pyl-leucylamido-(4-guanidino)butane; f.p.l.c., fast pro- al. (1977), and further work by Ryley (1979), tein liquid chromatography (Pharmacia system); [I]t, Sasaki's group (Sasaki et al., 1981 ; Taniguchi et al., total inhibitor concentration; Ki, dissociation constant 1981) and Jarvinen (1979) showed that the for inhibitor; Ki(app), apparent dissociation constant for protein inhibitor uncorrected for competition by substrate; [S], occurs in relatively low-Mr and high-Mr forms in substrate concentration; SDS, sodium dodecyl sulphate; plasma, with C2 and al electrophoretic mobilities v0, rate; vi, rate in the presence of inhibitor. respectively. Jarvinen (1979) described the purifi- * Present address: Department of Chemistry, Vassar cation ofthe two forms by use of affinity chromato- College, Poughkeepsie, NY 12601, U.S.A. graphy on a form of immobilized papain. Vol. 221 446 A. D. Gounaris, M. A. Brown and A. J. Barrett Experimental NMec as substrate at pH 6.0, and cathepsin H was Materials assayed with Arg-NMec as substrate at pH6.5 Plasma was from blood supplied by a local (Barrett & Kirschke, 1981; Barrett et al., 1982). blood-transfusion centre, and contained acid Dipeptidyl peptidase I was assayed with Gly-Phe- citrate/dextrose anticoagulant as described pre- NMec (10 gM) in 0.1OM-sodium phosphate buffer, viously (Barrett et al., 1979). Volumes of plasma pH 6.5, containing 1 mM-EDTA, 50mM-NaCl and given in the text have been corrected for dilution 1 mM-dithiothreitol. Trypsin was assayed with Bz- by the anticoagulant. DL-Arg-NPhNO2 as substrate, thrombin with Cm-papain-Sepharose (0.6mg of papain/g wet Boc-Val-Pro-Arg-NMec, and plasma kallikrein wt. ofgel) was prepared as described by Anastasi et with Z-Phe-Arg-NMec, all as described by Nagase al. (1983), and Cm-chymopapain-Sepharose was & Barrett (1981). prepared in an analogous manner. Chymopapain Quantification of inhibitory activity was partially purified from latex of Carica papaya by a salt fractionation procedure based on that of The procedure used for titration of aCPI with Brocklehurst et al. (1981). Papain was isolated papain was one previously described for cystatin, from Carica papaya latex by a modification of the in which the substrate was Z-Phe-Arg-NMec method of Baines & Brocklehurst (1979). Ficin (Anastasi et al., 1983). (twice-crystallized) was from Sigma (London) Ki values were determined by use of continuous Chemical Co. Bromelain (stem) was the crystalline fluorimetric assays in a Perkin-Elmer LS-3 suspension from Boehringer Corp. (London) Ltd. spectrofluorimeter standardized with 0.2 MM- Human cathepsins B and H were isolated as aminomethylcoumarin for readings at A, 360nm described by Schwartz & Barrett (1980). Human and Aexc. 460nm. The substrates (as used in assays: cathepsin L was purified from a homogenate of see above) were at 10Mm final concentration, and human liver by acid treatment, acetone fractiona- all measurements were made with less than 2% tion, gel chromatography and f.p.l.c. ion-exchange hydrolysis. For cathepsin B and cathepsin H, Km chromatography (G. D. J. Green & A. J. Barrett, was known to be much greater than 10,UM, with the unpublished work). Human plasma kallikrein respective substrates (Barrett & Kirschke, 1981). and antiserum against it (which also reacts with For human cathepsin L, Km for Z-Phe-Arg-NMec prokallikrein) were available in the laboratory was found to be 2 pM by the method of Wilkinson (Nagase & Barrett, 1981), as were the human liver (1961) (A. J. Barrett, unpublished work). For cysteine-proteinase inhibitors cystatin A and papain and dipeptidyl peptidase I, v0/[S] was cystatin B, and antisera against them (Green et al., constant in the range 5-20 M substrate, so it was 1984). Bovine spleen dipeptidyl peptidase I, kindly concluded that Km > 10Mm. The concentrations of given by Dr. J. K. McDonald (Medical University the enzymes during the assay were approx. 0.02nM of South Carolina, Charleston, SC, U.S.A.), was (cathepsin B), 0.1 nm (human cathepsin L), 0.1 nM further purified by f.p.l.c. Neuraminidase (Vibrio (cathepsin H), 0.01 nM (papain) and <0.1 nM cholerae; 500 units/ml) was from Koch-Light (dipeptidyl peptidase I). Laboratories. Bovine trypsin (type XII) and bovine The method was as follows. The fluorimeter thrombin were from Sigma. cuvette and all solutions were pre-warmed to 30°C Samples of purified aCPI (mixed high-Mr and (20°C for cathepsin L). Into the cuvette was placed low-Mr forms) and anti-(aCPI) serum for compari- 40ul of stock enzyme solution and 40M1 of 100mM- sons were kindly given by Dr. H. C. Ryley (Welsh dithiothreitol. A 1-2 min period was allowed for National School of Medicine, Cardiff, U.K.). activation, and then 3.84ml of the appropriate Antiserum to papain was kindly given by Dr. E. assay buffer and 40pl of 1 mm substrate solution in Shapira, The Children's Memorial Hospital, Chi- dimethyl sulphoxide were thoroughly mixed in. cago, IL, U.S.A. Antisera against antithrombin Once a stable reaction rate had been recorded, the III, al-antichymotrypsin and haptoglobin were inhibitor was added in 40Ml and the whole mixed purchased from Behringwerke A.G. The non-ionic well. The reaction rate was followed for up to detergent Brij 35 was purchased from BDH 60min as it relaxed progressively to the new linear Chemicals, and Ultrogel AcA-44 from LKB rate corresponding to the concentration of enzyme Instruments. Di-isopropyl phosphorofluoridate in equilibrium with the enzyme-inhibitor com- was from Sigma, and Pro-Phe-Arg-CH2CI was a plex. Control experiments were made to detect gift from Dr. E. N. Shaw, Brookhaven National spontaneous decay of enzymic activity during the Laboratory, Upton, NY, U.S.A. period of the experiment. Typically, data were obtained for five inhibitor concentrations. Ki Enzyme assays values were determined from re-plots of the form Papain, bromelain, cathepsin B and cathepsin L [I],/(1-vi/vo) versus vo/vi (Henderson, 1972). When were assayed fluorimetrically with Z-Phe-Arg- experiments were made with [S] < Kn, corrections 1984 a-Cysteine proteinase inhibitor 447 were made by use of the relationship Ki ing 0.5M-NaCl and 0.1% Brij 35. The column was = Ki(app.)/(1 + [S]I/K) for simple competition. washed with 500ml of the citrate buffer followed by about 2 litres of 0.05M-potassium phosphate Raising of antiserum buffer, pH8.5, also containing the NaCl and Brij A sheep was injected on three occasions at 2- 35, to lower the A280 of the effluent to less than week intervals; on each occasion, 1 mg of purified 0.05. The inhibitor was eluted with 0.05M-potas- low-Mr aCPI in 1 ml was emulsified with an equal sium phosphate/NaOH, pH 11.5, also containing volume of Freund's adjuvant, and injected intra- the NaCl and Brij 35.

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