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Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography
J. Biochem. Biophys. Methods 49 Ž2001. 491–505 www.elsevier.comrlocaterjbbm
Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography Gunther Jahreis, Klaus Peters ) , Heidrun Kirschke ¨ Institute of Physiological Chemistry, Faculty of Medicine, Martin Luther UniÕersity, Hollystrasse 1, D-06097 Halle (Saale), Germany
Abstract Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose ŽAMK-Sepharose. is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase ŽAAP. from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells ŽMP-1.. Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Alanyl aminopeptidase; Myeloma cell proteinase-1; Subtilisins; Cathepsins; Methyl ketones; Chloromethyl ketones; Antibody purification
1. Introduction Affinity chromatography can be a very effective step in the purification procedures of proteolytic enzymes. Most of the reviewed ligands Ždyes, antibiotics, natural protease AbbreÕiations: AAP, Alanyl aminopeptidase; Aca, ´-Aminocaproic acid; AH-Sepharose, AminohexylSepharose 4B; AMK-Sepharose, Alanine methyl ketone-AH-Sepharose 4B; Bz, Benzoyl; CK, Chloromethyl ketone Ž –CH 2 Cl.; DEAE-Sepharose, Diethylaminoethyl-Sepharose; DPP IV, Dipeptidyl aminopeptidase IV; EDTA, Ethylene diamine teraacetic acid; K i , Inhibition constant; k inact , 1st order rate constant of inhibition; MCA, Methylcoumarylamide; MK, Methyl ketone Ž –CH 3 .; MP-1, Myeloma cell proteinase-1; bNA, 2-Naphthylamide; pNA, 4-Nitroanilide; NEM, N-Ethylmorpholine; PAGE, Polyacrylamide gel electrophoresis; Suc, Succinyl; THF, Tetrahydrofuran; Z, Benzyloxycarbonyl. ) Corresponding author. Tel.: q49-345-552-5693; fax: q49-345-552-7030. E-mail address: [email protected] ŽK. Peters.. 0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 2 1 6 - 0
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inhibitors, etc.. are relatively nonspecific w1x. Synthetic substrate-analogous amino acid and peptidyl inhibitors can be adapted to the specificity of the peptidases to be purified. Therefore, we prefer them as selective affinity ligands. In 1987, we introduced amino acid methyl ketones w2x and, in 1990, peptidyl methyl ketones w3x as ligands in affinity chromatography. Here, we describe the use of a Sepharose with spacer-bound alanine methyl ketone as affinity ligand for the purification of a bacterial membrane-bound AAP w4x. Furthermore, we describe the purification of a tumor cell proteinase ŽMP-1. from the cytoplasmic membrane of myeloma cells w5,6x with Arginine-Agarose, containing C 12 -spacer arms, as support. Peptidyl methyl ketones are reversible inhibitors of serine and cysteine proteases w7–9x, and they are inert towards enzymatic degradation. Therefore, we used them as ligands for the affinity purification of thermitase and cathepsins B, L, and S. Among other techniques, affinity chromatography has also been applied in the purification of antibodies w10x. The antigens, used as ligands, were often nonspecifically bound to the matrix, and this causes deformation of the antigen molecules and steric problems w11x. An oriented immobilization of the antigen on the support should be preferable. We present spacer-bound peptidyl chloromethyl ketones as tools for a specific and oriented immobilization of proteinases as antigens. Via the active site, immobilized cathepsin B was used to purify antibodies against human cathepsin B.
2. Experimental 2.1. Materials 2.1.1. Substrates Ala-4-nitroanilide hydrochloride ŽAla-pNA HCl., Benzoyl-Arg-2-naphthylamide ŽBzArg-bNA., Benzyloxycarbonyl-Phe-Arg-methylcoumarylamide ŽZ-Phe-Arg-MCA. and Z-Arg-Arg-MCA were purchased from Bachem ŽHeidelberg, Germany.. Pro-Ala-pNA. HCl was synthesized according to Ref. w7x, Z-Val-Val-Arg-MCA according to Ref. w12x and Succinyl-Ala-Ala-Phe-pNA ŽSuc-Ala-Ala-Phe-pNA. according to Ref. w13x. 2.1.2. Inhibitors Z-Ala-chloromethyl ketone ŽZ-AlaCK. and Z-Phe-AlaCK were synthesized as previously described by Ref. w8x. The peptidyl methyl ketones ŽMK. were prepared according to the procedures of Refs. w14,15x. 2.1.3. Supports Aminohexyl-Sepharose 4B ŽAH-Sepharose 4B. was obtained from Pharmacia ŽUppsala, Sweden. and Arginine-Agarose ŽC 12 -spacer. from Sigma ŽDeisenhofen, Germany.. Divicell, a cellulose-based support, activated with 5-norbornene-2,3-dicarboximido carbonochloridate Ž30–34 mmol of activated hydroxyl groups per ml sedimented gel., was a gift from Dr. H.-F. Boeden ŽCentral Institute of Molecular Biology, Berlin, Germany. ŽArzneimittelwerke, Leipzig, Germany.. and Dr. R. Muller ¨
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2.1.4. Enzymes AAP from Acinetobacter calcoaceticus was prepared according to Ref. w4x and MP-1 according to Ref. w5x. Subtilisins BPNX and Carlsberg were purchased from Boehringer ŽMannheim, Germany.. Subtilisin DY was a gift from Prof. N.C. Genov ŽBulgarian Academy of Sciences, Sofia, Bulgaria.. A concentrated supernatant of culture medium from Thermoactinomyces Õulgaris as well as pure thermitase, purified by isoelectric ŽHumboldt University, Berlin, Germany.. focusing, were a gift from Prof. W. Hohne ¨ Pure thermitase, purified by adsorption on porous glass beads, was a gift from Dr. U. Kettmann ŽMartin Luther University, Halle, Germany.. Cathepsin B from human liver and rat liver, respectively, were used as crude enzyme preparations after extraction, autolysis, acetone fractionation and DEAE-Cellulose ŽDiethylaminoethyl-Cellulose. chromatography according to Ref. w16x. 2.1.5. Antibodies The immunization scheme for goats was the same as described by Ref. w17x for rabbits. Immunoglobulins were prepared by the modified caprylic acid method w17x. 2.1.6. Other materials All chemicals were trade products with a high purity. The Diaflo ultrafiltration membranes YM-5, YM-10, and YM-30, used for the Amicon ultrafiltration systems, were purchased from Amicon ŽBeverly, MA, USA.. 2.2. Methods 2.2.1. Synthesis of AMK-sepharose 6X-N-ŽL-Alanine methyl ketone.-aminohexyl-Sepharose hydrochloride, AMK-Sepharose Ž5.: According to Scheme 1, Z-AlaCK Ž16 mg; 60 mmol., 2, was added to a suspension of AH-Sepharose 4B Ž1.5 g., 1, in 20 ml tetrahydrofuran ŽTHF.. N-Ethylmorpholine Ž10 ml; 79 mmol, NEM. was then added and the reaction mixture was stirred at 4 8C for 24 h. Product 3 was isolated by filtration, washed thoroughly with 100 ml THF and dried. Two milliliters HBrrglacial acetic acid Ž4 molrl. was added to the dry powder Ž1.5 g. of 3. The reaction was gently stirred at 4 8C. After 1 h, 30 ml diethyl ether was added, and the product 4 was separated by filtration. The solid was then washed with 50 ml ethylene glycol in water Ž10 vol.%. and, thereafter, with TrisrHCl buffer Ž0.05 molrl, pH 7.5. to yield compound 5. 2.2.2. Purification of the Acinetobacter alanyl aminopeptidase on AMK-sepharose A column Ž100 = 5 mm. of AMK-Sepharose Ž20–25 mmol AlaMK per g dry gel w4x. was equilibrated at 4 8C with TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100.. Then, 0.5 ml Triton X-100-containing membrane-protein extract Ž5.1 mg protein. w4x was applied to the column at a flow-rate of ca. 2 mlrh at 4 8C ŽFig. 1.. The fractionation was started after elution of ca. 3 ml. The column was then washed with 15 ml buffer. Gradient elution was carried out with increasing concentration of KCl Ž0–1.0 molrl. in
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Scheme 1. Synthesis of AMK-Sepharose as affinity chromatography matrix for purification of a membranebound AAP. 1: AH-Sepharose 4B, 2: Z-AlaCK, 5: AMK-Sepharose.
Fig. 1. Purification of the AAP from A. calcoaceticus by affinity chromatography on AMK-Sepharose. -'Absorbance at 280 nm Žprotein., -v- AAP activity ŽAla-pNA., -B- DPP IV activity ŽPro-Ala-pNA.; 0.5 ml Triton X-100-containing membrane protein extract was applied to the column Ž100=5 mm. at a flow-rate of ca. 2 mlrh at 4 8C. The fractionation was started after elution of ca. 3 ml. The gradient was carried out with increasing concentration of KCl in TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100..
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the TrisrHCl buffer. The AAP was eluted at 0.6 molrl KCl. The pooled fractions, containing pure Ala-pNA activity for AAP, were concentrated to ca. 4 ml Ž16 mgrml protein w18x., using an Amicon YM-30 membrane. Exopeptidase activities ŽFig. 1. were measured by incubation of 100-ml aliquotes of the eluent fractions with the chromogenic substrates Ž3 mmolrl. for 1 h at 37 8C and detection of the liberated 4-nitroaniline at l s 405 nm. Protein was assayed in the effluent at l s 280 nm. 2.2.3. Purification of the myeloma cell proteinase-1 on Arginine-Agarose A prepurified preparation of MP-1 Ž0.6 ml, 0.43 mg protein. w5x, after Sepharose 6B chromatography and concentration on an Amicon YM-30 membrane, was applied to a column Ž80 = 5 mm. of Arginine-Agarose ŽC 12 -spacer., equilibrated with TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100. at 4 8C. The flow-rate was 2 mlrh. The loaded column was then washed with 12 ml buffer for ca. 3 h. The bound MP-1 was eluted with increasing concentrations of NaCl Ž0–0.5 molrl. in the TrisrHCl buffer. Fractions Ž0.5 ml. were collected at a flow-rate of 2 mlrh. Protein was assayed in the effluent at l s 280 nm. MP-1 activity was detected by incubation of the eluent fractions Ž100 ml. with Bz-Arg-bNA Ž2 mmolrl. for 1 h at 37 8C ŽFig. 2a.. The enzymatically liberated 2-naphthylamine was measured at l s 340 nm. All fractions, containing Bz-Arg-bNA activity for MP-1, were pooled and concentrated to ca. 2.5 ml Ž38 mgrml protein w19x., using an Amicon YM-30 membrane. 2.2.4. Synthesis of DiÕicell-Aca-inhibitor affinity gels Activated Divicell gel was coupled with ´-aminocaproic acid ŽAca. in sodium tetraborate buffer Ž0.1 molrl, pH 8.0. to give support-spacer conjugates ŽDivicell-AcaOH. with 17–21 mmol carboxyl groups per ml sedimented gel. The N-deprotected peptidyl methyl ketones were coupled to the carboxyl groups of the spacer, using N-ethyl-N X-Ž3-dimethylamino-.propyl carbodiimide hydrochloride, at pH 4.5–5.0. Distilled water or, if the peptidyl derivative is insoluble in water, a dioxanrwater mixture was used as solvent. The gels were washed with 10 volumes of the gel volume with water, and then four times alternating with TrisrHCl buffer Ž0.1 molrl, pH 8.0, 0.5 molrl NaCl. and acetic acidracetate buffer Ž0.1 molrl, pH 4.0, 0.5 molrl NaCl.. All gels were stored in phosphate buffer Ž0.1 molrl, pH 6.0, 1.0 molrl NaCl, 0.02% NaN3 . at 4 8C. The affinity gels were characterized by amino acid analysis after total hydrolysis. The concentration of the immobilized ligands was determined to be between 12 and 17 mmolrml sedimented gel w15x. N-Deprotected peptidyl chloromethyl ketones were coupled to Divicell-Aca-OH using the same method. In the washing procedure, the time of exposure with TrisrHCl buffer Ž0.1 molrl, pH 7.5. was minimized. 2.2.5. Purification of thermitase on DiÕicell-Aca-Ala-Ala-PheMK A concentrated supernatant of culture medium of T. Õulgaris was prepurified by size-exclusion chromatography on Sephadex G-75 in ammonium acetate buffer Ž0.1 molrl, pH 7.5. at 4 8C. 65 ml Ž0.525 mg proteinrml. of the thermitase-containing fractions was applied to a column Ž6 = 50 mm. of Divicell-Aca-Ala-Ala-PheMK, equilibrated with the ammonium acetate buffer, at a flow-rate of 3 mlrh and at 4 8C.
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Fig. 2. Ža. Affinity chromatography of MP-1 from myeloma cells on Arginine-Agarose. -v- Absorbance 280 nm Žprotein., -B- MP-1 activity ŽBz-Arg-bNA.. A prepurified preparation of MP-1 was applied to the column Ž80=5 mm.. Subsequent elution of MP-1 was carried out with NaCl in TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100.. Žb. SDS polyacrylamide gel electrophoreses Ž12.5%, pH 8.8 w37x. of the purified MP-1 under reducing conditions with 0.1% SDS and 0.1% 2-mercaptoethanol, followed by Coomassie Blue staining. Lanes 2–5: purified MP-1, lanes 1 and 6: marker proteins ŽkDa.: 96, phosphorylase; 55, glutamate dehydrogenase; 39, aldolase; 30, carbonic anhydrase; 21, trypsin inhibitor.
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After washing with ammonium acetate buffer Žcontaining additionally 0.5 mmolrl CaCl 2 ., TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 . and, finally, addition of NaCl Ž1.0 mmolrl. to the TrisrHCl buffer, the protease was eluted with 2-propanol Ž40 vol.%., added to the TrisrHCl buffer, containing CaCl 2 and NaCl. During the washing procedure, the flow-rate was 12 mlrh. Elution was carried out at a flow-rate of 5 mlrh. The protein concentrations were determined by the Lowry et al. w18x method. 2.2.6. Purification of human cathepsin B on DiÕicell-Aca-Phe-PheMK A crude preparation of cathepsin B from human liver Ž5.5 ml, 1.06 mg proteinrml. in acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. was activated with dithioerythritol Ž10 mmolrl. and EDTA Ž10 mmolrl. and then slowly Žflow-rate, 3 mlrh. at 4 8C applied to a column Ž40 = 6 mm. of Divicell-Aca-Phe-PheMK. The affinity gel was washed with the same acetic acidracetate buffer, containing additionally NaCl Ž1.0 molrl., at a flow-rate of 5 mlrh. The bound enzyme was eluted with 40 vol.% ethylene glycol and 1 vol.% 2-mercaptoethanol in the washing buffer Žflow-rate, 4 mlrh.. All fractions, containing the same buffer, were pooled, and the pooled fractions were concentrated to ca. 2 ml, using an Amicon YM-5 membrane. The activity of cathepsin B was monitored with Z-Phe-Arg-MCA. In experiments with mixtures of cathepsins B and L, Z-Arg-Arg-MCA was additionally used and the enzymes discriminated as described in Ref. w16x. The enzymatically liberated methylcoumaryl amine was measured at lex s 380 nm, lem s 460 nm. The activity of cathepsin S in other experiments was monitored with Z-Val-Val-Arg-MCA. 2.2.7. Purification of cathepsin B-antibodies on an affinity matrix with oriented-immobilized cathepsin B 2.2.7.1. Immobilization of cathepsin B. A crude preparation of cathepsin B from rat liver was activated with dithioerythritol Ž10 mmolrl. in the presence of EDTA Ž10 mmolrl. and mixed with 10 ml affinity gel Divicell-Aca-Phe-AlaCK Žsediment, 3.5 ml. in acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. at room temperature. The suspension was shaken until the enzyme activity in the supernatant decreased to a negligible value. The gel was washed with distilled water and four times alternately with acetic acidracetate buffer Ž0.1 molrl, pH 5.0. and TrisrHCl buffer Ž0.1 molrl, pH 8.2., and then stored in the TrisrHCl buffer, containing additionally 0.02% NaN3 . 2.2.7.2. Preparation of the cathepsin B antibodies. An immunoglobulin fraction Ž60 mg proteinr2 ml. from goat antiserum against human cathepsin B in TrisrHCl buffer Ž0.02 molrl, pH 8.2. was slowly applied to a column Ž58 = 6 mm. of Divicell-Aca-Ala-Phecathepsin B at room temperature. After 30 min, the gel was washed with 10 ml of the TrisrHCl buffer and then with 10 ml of TrisrHCl buffer, containing additionally 1.0 molrl KCl. The antibodies to cathepsin B were eluted with 15 ml glycinerHCl buffer Ž0.2 molrl, pH 2.2. and collected in the same volume of TrisrHCl buffer Ž0.5 molrl, pH 8.5.. All fractions, containing the same buffer, were pooled and concentrated to ca. 1 ml, using an Amicon YM-10 membrane.
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3. Results and discussion 3.1. Purification of alanyl aminopeptidase from A. calcoaceticus The isolation of membrane-bound exopeptidases from a detergent-containing membrane protein solution by size-exclusion or ion-exchange chromatography is less successful. Affinity chromatography offers the possibility of enzyme-selective adsorption and subsequent recovery of the enzyme from an immobilized enzyme-specific ligand. The merits of immobilized inhibitors, dyes, or antibiotics for protease purification are explained by Ref. w1x. Active-site-directed reversible inhibitors of proteases are useful reagents in protein purification. Amino acid chloromethyl ketones and methyl ketones have been shown to be potent reversible inhibitors of soluble w9x and membrane-bound aminopeptidases w20x. Alanine methyl ketone is a strong competitive inhibitor Ž K i ( 10y6 molrl. w21x of Acinetobacter AAP. Alanine methyl ketone was chosen for immobilization because it binds to the aminopeptidase more strongly than the natural ligand alanine. Therefore, we synthesized alanine-methyl-ketone-modified AH-Sepharose ŽScheme 1. as affinity support. Alanine chloromethyl ketone was covalently bound to the AH-Sepharose by a linkage between the v-amino group of the alkyl side chain of the AH-Sepharose and the chloromethyl function of AlaCK w2x. This matrix was able to bind Acinetobacter AAP specifically and selectively ŽFig. 1.. The AAP was tightly adsorbed at pH 7.5 and 4 8C to the AMK-Sepharose matrix. Furthermore, Pro-Ala-pNA activity was bound weaker to the affinity adsorbent than the AAP. Non-adsorbed proteins were washed out with TrisrHCl buffer at pH 7.5. The specific complex of the AAP with the immobilized alanine methyl ketone could be decomposed with KCl. With an increasing KCl gradient, the Pro-Ala-pNA activity ŽDPP IV. was eluted as first peak at ca. 0.20 molrl KCl ŽFig. 1.. The AAP peak was eluted at 0.6 molrl KCl in good yield Ž78% of applied Ala-pNA activity.. It is suggested that the AAP, a SH-dependent exopeptidase of 212 kDa w4x, forms a thiohemiacetal in the active site of AAP with the carbonyl function of the immobilized alanine methyl ketone ligand at the affinity matrix. The mechanism is likely to be the same as that shown for the inhibition of AAP by soluble alanine methyl ketone w20x. Endopeptidases were not bound by AMK-Sepharose. The gel was stable and the AMK-ligand was not hydrolyzed by enzymes. The homogeneity of the AAP from A. calcoaceticus was confirmed by polyacrylamide gel electrophoresis w4x. From these results, it is evident that affinity chromatography is a very useful method for the purification of detergent solubilized exopeptidases. 3.2. Purification of myeloma cell proteinase The usual procedures of protein separation and purification are based on the relatively small differences in shape, size, and charge of water-soluble proteins in the mixture. In contrast, detergent-solubilized membrane-bound proteinases exist in a detergent-protein mixed micelle form. A good approach to the purification of micellanous associated membrane proteases employs affinity chromatography on proteinase-selective ligands.
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The MP-1 is a membrane-bound proteinase from the plasma membrane of myeloma cells. The enzyme was identified as a trypsin-like peptidase. MP-1 cleaves the substrate Bz-Arg-bNA optimally at pH 7.5 at 37 8C w6x. After treatment of the plasma membrane fragments with Triton X-100, a soluble membrane protein mixture, containing MP-1, was obtained. It was not possible to purify MP-1 from this protein mixture by size-exclusion or ion-exchange chromatography. MP-1 was contaminated with membrane-type matrix metalloproteinases w22x, cathepsin, and serine protease activities w23x. Arginine-Agarose with C 12 -spacer chains was shown to be an effective substrate-like affinity sorbent for MP-1 in contrast to Arginine-Agarose without spacer, which did not adsorb MP-1. Fig. 2a represents the affinity chromatography of the MP-1 from a Triton X-100-containing membrane protein extract, pretreated on a Sepharose 6B column. The contaminated MP-1 solution was applied to the affinity column. After washing off non-adsorbed membrane proteins with TrisrHCl buffer, the MP-1 was eluted with a linear gradient of NaCl in a yield of 89% of the applied activity and in very high purity. The homogeneity of MP-1 was demonstrated by polyacrylamide gel electrophoresis ŽFig. 2b. and Western blot analysis. MP-1 was found to consist of four identical subunits with molecular weights of 64 kDa ŽSDS-PAGE.. Arginine-specific proteinases, like cathepsins or plasminogen activator, were detectable only in the washing buffer eluate but not in the gradient fractions. The desorption of MP-1 was also successful in the presence of Triton X-100 concentrations of 1% in the buffer. 3.3. Affinity purification of thermitase and related proteases Peptidyl methyl ketones as ligands of proteinases are effective competitive inhibitors for some subtilisins and related proteinases, especially thermitase w14,24,25x, and, therefore, suitable as ligands in affinity chromatography. According to the specificity of the subtilisins and subtilisin-related enzymes, the most efficient peptidyl methyl ketone was found to be Z-Ala-Ala-PheMK with an inhibition constant Ž K i . of 3.0 = 10y7 molrl for thermitase, 9.4 = 10y6 molrl for subtilisin Carlsberg, 3.1 = 10y5 molrl for subtilisin DY, 1.3 = 10y5 molrl for proteinase K, and 1.3 = 10y4 molrl for subtilisin BPNX , whereas a-chymotrypsin was inhibited only with a K i of 1.1 = 10y3 molrl w13,25x. It has been shown that the subtilisins Carlsberg and DY as well as proteinase K and thermitase are quantitatively adsorbed on affinity gels with Aca as spacer and Ala 2-PheMK as ligand w3,15x. As support, Divicell is preferable to Sepharose since Divicell has advantages with respect to the binding capacity and to the stability of the gel, which causes to the very stable urethane bonding between support and spacer w26,27x. The elution pattern of a Sephadex G-75-prepurified thermitase preparation from Divicell-Aca-Ala-Ala-PheMK is shown in Fig. 3. The adsorbed thermitase was eluted in a sharp peak by of 2-propanol Ž40 vol.%.. 2-Propanol reduces dramatically the enzymatic activity. This effect, however, is completely reversible and has the advantage of suppressing autolysis, which otherwise destroy considerable amounts of the active enzyme w15x. The specific activity was increased from 1.3 to 3.0 mkatrmg protein by the affinity chromatography. Fig. 4 shows the electrophoresis pattern of the Sephadex G-75-prepurified thermitase and of the purified thermitase after affinity chromatography in comparison with preparations, purified by two other methods w28,29x. Divicell-Aca-
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Fig. 3. Affinity chromatography of thermitase on Divicell-Aca-Ala-Ala-PheMK. -v- Absorbance at 280 nm Žprotein., -B- relative activity against Suc-Ala-Ala-Phe-pNA: activity of the eluateractivity of the applied solution. Dotted lines indicate the change of the eluent. 1: applied solution—65 ml Sephadex G-75-prepurified thermitase Žprotein: 0.525 mgrml. in ammonium acetate buffer Ž0.1 molrl, pH 7.5.; 2: ammonium acetate buffer Ž0.1 molrl, pH 7.5, 0.5 mmolrl CaCl 2 .; 3: TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 .; 4: TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 , 1.0 molrl NaCl.; 5: TrisrHCl buffer Ž0.1 molrl; pH 7.8, 0.5 mmolrl CaCl 2 , 1.0 molrl NaCl, 40 vol.% 2-propanol.. Column 50=6 mm; flow-rates, 1: 3 mlrh; 2–4: 12 mlrh; 5: 5 mlrh.
Ala-Ala-PheMK proved very suitable for purification of thermitase, although the immobilized inhibitor Ž K i ca. 3.5 = 10y6 molrl. was ca. 100 times less potent than Z-Ala-Ala-PheMK and ca. 10 times less potent than Ac-Aca-Ala-Ala-PheMK, mimicking the spacer-ligand unit w15x. Subtilisins and proteinase K have been eluated from Divicell-Aca-Ala-Ala-PheMK with lower concentrations of 2-propanol than thermitase according to their decreased inhibition by Z-Ala-Ala-PheMK. 3.4. Affinity purification of the cathepsins B, L and S Since peptidyl methyl ketones are inhibitors not only of a number of serine proteases, but also of cysteine proteases Žpapain, cathepsins B, L, and S w12,30,31x., we tried to find useful affinity matrices with peptidyl methyl ketones as ligands and favorable chromatographical conditions for the purification of the cathepsins mentioned. DivicellAca-Phe-AlaMK and Divicell-Aca-Phe-PheMK were tested as affinity media for the purification of cathepsins B and L. Only Divicell-Aca-Phe-PheMK proved a suitable
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Fig. 4. SDS polyacrylamide gel electrophoresis of thermitase. Conditions: 0.1% SDS, 15% polyacrylamide gel ŽpH 8.8., Coomassie Blue staining. In order to prevent autolysis during the boiling with SDS, thermitase was denaturated with 20% trichloroacetic acid ŽLanes 1 and 2. and incubated with Z-Ala-Ala-PheCK for 90 min. ŽLanes 3–5., respectively, before treatment with SDS. Lanes: 1, crude product; 2, prepurified thermitase after size-exclusion chromatography with Sephadex G-75; 3, after affinity chromatography; 4, after isoelectric focusing; 5, after adsorption on porous glass beads; 6, marker proteins ŽkDa.: 96, phosphorylase; 66, albumin; 45, ovalbumin; 30, carbonic anhydrase; 21, trypsin inhibitor; 14.4, a-lactalbumin.
affinity gel for purifying the enzymes. A similar elution profile of cathepsin B, as shown in Fig. 5, was obtained with cathepsin L. Buffer 3 eluted 62% of the applied activity of cathepsin B and 11% of the applied protein ŽFig. 5.. The total recovery was 69% of the protein and 85% of the activity. Western blot analysis with polyclonal antibodies to human cathepsin B and human cystatins A and B revealed the presence of different concentrations of cathepsin B in all fractions, shown in Fig. 5, and, in addition, complexes of cathepsin B with the cystatins. Peptidyl diazomethyl ketones have often been described as selective irreversible inhibitors of cysteine proteases w32x, but they also inhibit some serine proteases w33x. The inhibitor, which is most discriminating between cathepsin B and cathepsin L, is Z-Phe-TyrŽO-t-Bu.CHN2 w34x. The inactivation rate k inactrK i for cathepsin L is 1.2 = 10 7 My1 miny1 , for cathepsin B, 6.2 = 10 2 My1 miny1 , leading to a relative inactivation rate LrB of ca. 20.000 w34x. These kinetic data suggested that the dipeptidyl methyl ketone Phe-TyrŽO-t-Bu.MK could be a suitable affinity ligand for separating cathepsins B and L from a mixture of the enzymes. However, use of Divicell-Aca-Phe-TyrŽO-tBu.MK for the separation of cathepsin B and L did not come up to our expectations. Only an enrichment of each enzyme, but not a complete separation, could be achieved. Ca. 75% of the applied cathepsin B activity was eluted without retention, whereas cathepsin L was almost completely bound to the affinity gel and eluted by additions of NaCl, ethylene glycol, and 2-mercaptoethanol to the acetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. used.
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Fig. 5. Affinity chromatography of human cathepsin B on Divicell-Aca-Phe-PheMK. -v- Absorbance 278 nm Žprotein., -B- activity against Z-Phe-Arg-MCA. Dotted lines indicate the change of the eluent. 1: applied solution—5.5 ml crude cathepsin B Žprotein: 1.06 mgrml. in standard acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA.; 2: standard buffer, containing NaCl Ž1.0 molrl.; 3: standard buffer, containing NaCl Ž1.0 molrl., ethylene glycol Ž40 vol.%., and 2-mercaptoethanol Ž1 vol.%.; 4: the affinity gel was left overnight in buffer 3. Column: 40=6 mm; flow-rates, 1: 3 mlrh; 2, 3: 5 mlrh, 4: 4 mlrh.
Inhibition of cathepsin S by Z-Val-Val-PheMK has a K i value of 1.8 = 10y6 molrl w12x. Therefore, we tested Divicell-Aca-Val-Val-PheMK as affinity support for cathepsin S from bovine spleen. Cathepsin S bound completely at pH 7.0 Ž0.1 molrl phosphate buffer. to the gel. After washing to remove contaminating proteins, the enzyme was eluted at pH 5.0 Ž0.1 molrl acetic acidracetate buffer. with additions of NaCl, 2-propanol and 2-mercaptoethanol to the buffer. 3.5. Purification of anti-cathepsin B on affinity-immobilized cathepsin B In the affinity purification of antibodies, affinity supports have been used mainly where the antigen molecules are nonspecifically bound to the spacer arms or directly to the matrix via amino or carboxyl groups at the surface of the antigen w11x. The consequence of such a coupling is that each of the antigen molecules is nonspecifically and in a different manner immobilized. It is assumed that a large number of antigen molecules are linked to the support via several functional groups, causing deformation of the molecules. Problems with respect to the steric accessibility of the antigen may also
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occur w11x. Therefore, oriented immobilization of proteases, acting as antigens, has the following advantages: Ža. all antigen molecules are immobilized in the same manner via one center, Žb. no significant deformation occurs, Žc. all molecules present the same epitopes to the antibodies, Žd. the affinant often shows an increased stability. We propose to use specific spacer-bound peptidyl chloromethyl ketones for the covalent immobilization of proteases via their active site since chloromethyl ketones react spontaneously with cystein proteases by S-alkylation w35x and with serine proteases by N-alkylation w36x, respectively, of their catalytic sites. The formed modified matrix, consisting of support, spacer, peptide chain and protease Žantigen., is an affinity sorbent for antibodies to the immobilized protease. We synthesized Divicell-Aca-Phe-AlaCK for the irreversible binding of cathepsin B. The immunoglobulin fraction of an antiserum from goat against human cathepsin B was applied in TrisrHCl buffer ŽpH 8.2. to an affinity column of Divicell-Aca-Phe-AlaCH 2-cathepsin B. Ca. 42% and 11%, respectively, of the applied protein was washed out in two steps. The antibodies to cathepsin B Žca. 15%. were eluted at pH 2.2 with a glycinerHCl buffer, as revealed by Western blots with cathepsin B. Anti-cathepsin B could not be detected in any other fraction. This method has the advantage that crude preparations may serve as a source of proteases to be immobilized because of selective binding of the enzyme. It is to assume that most of the epitopes at the surface of the antigen-protease are not deformed, and, thus, the antibodies could bind in an optimal manner and could be obtained in maximal yield.
References w1x Ibrahim-Granet O, Bertrand O. Separation of proteases: old and new approaches. J Chromatogr, B: Biomed Sci Appl 1996;684:239–63. w2x Jahreis G, Aurich H. Synthese einer Alaninmethylketon-modifizierten AH-Sepharose zur affinitats¨ chromatographischen Reinigung einer bakteriellen Alaninaminopeptidase. Pharmazie 1987;42:270. w3x Peters K, Fittkau S. Affinitatschromatographie von Proteasen mit Peptidmethylketonen als Liganden. ¨ Biomed Biochim Acta 1990;49:173–8. w4x Jahreis G, Sorger H, Aurich H. Eine membrangebundene Alaninaminopeptidase aus Acinetobacter calcoaceticus: 1. Isolierung und Reinigung des Enzyms. Biomed Biochim Acta 1989;48:617–24. w5x Jahreis G, Preissner A, Weber E. A new growth releated endopeptidase from myeloma cells. Biol Chem Hoppe-Seyler 1994;375:58. w6x Jahreis G, Weber E, Lutz D, Preissner A. Characterization of a new endopeptidase from myeloma cells. Biol Chem Hoppe-Seyler 1995;376:S111. w7x Heins J, Welker P, Schonlein C, Born I, Hartrodt B, Neubert K, et al. Mechanism of proline-specific ¨ proteinases: substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from FlaÕobacterium meningosepticum. Biochim Biophys Acta 1988;954:161–9. w8x Jahreis G, Smalla K, Fittkau S. Thermitase—eine thermostabile Serinprotease: 2. Synthese von substratanalogen Peptidchlormethylketonen als potentielle irreversible Enzyminhibitoren. J Prakt Chem 1984;326:41–7. w9x Kettner C, Glover GI, Prescott JM. Kinetics of inhibition of Aeromonas aminopeptidase by leucine methyl ketone derivatives. Arch Biochem Biophys 1974;165:739–43. w10x Nau DR. Chromatographic methods for antibody purification and analysis. BioChromatography 1989; 4:4–17. w11x Turkova J. Oriented immobilization of biologically active proteins as tool for revealing protein interactions and function. J Chromatogr 1999;722:11–31.
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w12x Bromme D, Steinert A, Friebe S, Fittkau S, Wiederanders B, Kirschke H. The specificity of bovine spleen ¨ cathepsin S. A comparison with rat liver cathepsins L and B. Biochem J 1989;264:475–81. X w13x Bromme D, Fittkau S. A new substrate and two inhibitors applicable for thermitase, subtilisin BPN and ¨ a-chymotrypsin. Comparison of the kinetic parameters with customary substrates and inhibitors. Biomed Biochim Acta 1985;44:1089–94. w14x Fittkau S, Jahreis G. Synthese von N-Acyl-peptidmethylketonen als reversible Enzyminhibitoren. J Prakt Chem 1984;326:48–53. w15x Peters K, Steinert A, Strohl ¨ D, Fittkau S. Peptidyl methyl ketones as ligands in affinity chromatography of serine and cysteine proteases. J Chromatogr 1993;648:91–9. w16x Barrett AJ, Kirschke H. Cathepsin B, cathepsin H, and cathepsin L. Methods Enzymol 1981;80:535–61. w17x Wiederanders B, Kirschke H. Antibodies to the rat liver cathepsins: characterization and use for the identification of enzyme precursors. Biomed Biochim Acta 1986;45:1421–31. w18x Lowry OH, Rosebrough NH, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265–75. w19x Vera JC. Measurement of microgram quantities of protein by a generally applicable turbitimetric procedure. Anal Biochem 1988;174:187–96. w20x Jahreis G, Aurich H. Eine membrangebundene Alanylaminopeptidase aus Acinetobacter calcoaceticus: 4. Aminosaurechlormethylketone als pH-abhangige substratanaloge Cysteinprotease-Inhibitoren. Biomed ¨ ¨ Biochim Acta 1991;50:1043–9. w21x Jahreis G, Fittkau S, Aurich H. a-Aminomethylketone als Inhibitoren einer membrangebundenen Alaninaminopeptidase. Biomed Biochim Acta 1987;46:683–6. w22x Pei D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J Biol Chem 1999;274:8925–32. w23x Bjorquist P, Brohlin M, Ehnebom J, Ericsson M, Kristiansen C, Pohl G, et al. Plasminogen activator ¨ inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator. Biochim Biophys Acta 1994;1209:191–202. w24x Fittkau S, Smalla C, Pauli D. Thermitase—eine thermostabile Serinprotease: IV. Kinetische Untersuchungen der Bindung von N-Acylpeptidketonen als substratanaloge Inhibitoren. Biomed Biochim Acta 1984;43:887–99. w25x Peters K, Pauli D, Hache H, Boteva RN, Genov NC, Fittkau S. Subtilisin DY-Kinetic characterization and comparison with related proteinases. Curr Microbiol 1989;18:171–7. w26x Boeden HF, Pommerening K, Becker M, Rupprich C, Holtzhauer M, Loth F, et al. Bead cellulose derivatives as supports for immobilization and chromatographic purification of proteins. J Chromatogr 1991;552:389–414. w27x Baeseler M, Boeden HF, Koelsch R, Lasch J. Cellulose beads: a weak leaking affinity support. J Chromatogr 1992;589:93–100. w28x Frommel C, Hausdorf G, Hohne WE, Behnke U, Rutloff H. Charakterisierung einer Protease aus ¨ ¨ Thermoactinomyces Õulgaris ŽThermitase.: 2. Einschritt-Feinreinigung und proteinchemische Charakterisierung. Acta Biol Med Ger 1978;37:1193–204. w29x Kleine R, Rothe U, Kettmann U, Schelle H. Purification, properties, and application of thermitase, a microbial serine-protease from Thermoactinomyces Õulgaris. In: Turk V, Vitale LJ, editors. Proteinases and their inhibitors. Oxford: Pergamon; 1981. p. 201–12. w30x Bromme D, Bartels B, Peters K, Kirschke H, Fittkau S. The use of peptide methyl ketones as inhibitors of ¨ proteinases, peptides. In: Penke B, Torok ¨ ¨ A, editors. Peptides, Chemistry, biology, interactions with proteins. Berlin: Walter de Gruyter; 1988. p. 53–6. w31x Bromme D, Bartels B, Kirschke H, Fittkau S. Peptide methyl ketones as reversible inhibitors of cysteine ¨ proteases. J Enzyme Inhib 1989;3:13–21. w32x Shaw E, Green GDJ. Inactivation of thiol proteases with peptidyl diazomethyl ketones. Methods Enzymol 1981;80:820–6. w33x Ermer A, Baumann H, Steude G, Peters K, Fittkau S, Dolaschka P, et al. Peptide diazomethyl ketones are inhibitors of subtilisin-type serine proteases. J Enzyme Inhib 1990;4:35–42. w34x Kirschke H, Wikstrom P, Shaw E. Active center differences between cathepsins L and B: the S1 binding region. FEBS Lett 1988;228:128–30.
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w35x Drenth J, Kalk KH, Swen HM. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry 1976;15:3731–8. w36x Roberts ID, Alden RA, Birktoft J, Kraut J, Powers JC, Wilcox PE. An X-ray crystallographic study of the X binding of peptide chloromethyl ketone inhibitors to subtilisin BPN . Biochemistry 1972;11:2439–49. w37x O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem 1975;250:4007– 21.
Purification of soluble and membrane-bound proteases with substrate-analogous inhibitors by affinity chromatography Gunther Jahreis, Klaus Peters ) , Heidrun Kirschke ¨ Institute of Physiological Chemistry, Faculty of Medicine, Martin Luther UniÕersity, Hollystrasse 1, D-06097 Halle (Saale), Germany
Abstract Specific modified substrate-analogous amino acids and peptides have been used as affinity ligands in the affinity chromatography of proteases. Alanine methyl ketone-Sepharose ŽAMK-Sepharose. is introduced as affinity support for the purification of a bacterial alanyl aminopeptidase ŽAAP. from a membrane protein extract and Arginine-Agarose as support for the preparation of a membrane-bound proteinase of myeloma cells ŽMP-1.. Peptidyl methyl ketones as affinity ligands have been used to separate subtilisin enzymes and the cysteine proteases cathepsin B, L, and S. As a new type of ligands, spacer-bound peptidyl chloromethyl ketones are presented for a specific and oriented immobilization of proteinases. Oriented-immobilized cathepsin B was used to isolate antibodies against this enzyme. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Alanyl aminopeptidase; Myeloma cell proteinase-1; Subtilisins; Cathepsins; Methyl ketones; Chloromethyl ketones; Antibody purification
1. Introduction Affinity chromatography can be a very effective step in the purification procedures of proteolytic enzymes. Most of the reviewed ligands Ždyes, antibiotics, natural protease AbbreÕiations: AAP, Alanyl aminopeptidase; Aca, ´-Aminocaproic acid; AH-Sepharose, AminohexylSepharose 4B; AMK-Sepharose, Alanine methyl ketone-AH-Sepharose 4B; Bz, Benzoyl; CK, Chloromethyl ketone Ž –CH 2 Cl.; DEAE-Sepharose, Diethylaminoethyl-Sepharose; DPP IV, Dipeptidyl aminopeptidase IV; EDTA, Ethylene diamine teraacetic acid; K i , Inhibition constant; k inact , 1st order rate constant of inhibition; MCA, Methylcoumarylamide; MK, Methyl ketone Ž –CH 3 .; MP-1, Myeloma cell proteinase-1; bNA, 2-Naphthylamide; pNA, 4-Nitroanilide; NEM, N-Ethylmorpholine; PAGE, Polyacrylamide gel electrophoresis; Suc, Succinyl; THF, Tetrahydrofuran; Z, Benzyloxycarbonyl. ) Corresponding author. Tel.: q49-345-552-5693; fax: q49-345-552-7030. E-mail address: [email protected] ŽK. Peters.. 0165-022Xr01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 0 2 2 X Ž 0 1 . 0 0 2 1 6 - 0
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inhibitors, etc.. are relatively nonspecific w1x. Synthetic substrate-analogous amino acid and peptidyl inhibitors can be adapted to the specificity of the peptidases to be purified. Therefore, we prefer them as selective affinity ligands. In 1987, we introduced amino acid methyl ketones w2x and, in 1990, peptidyl methyl ketones w3x as ligands in affinity chromatography. Here, we describe the use of a Sepharose with spacer-bound alanine methyl ketone as affinity ligand for the purification of a bacterial membrane-bound AAP w4x. Furthermore, we describe the purification of a tumor cell proteinase ŽMP-1. from the cytoplasmic membrane of myeloma cells w5,6x with Arginine-Agarose, containing C 12 -spacer arms, as support. Peptidyl methyl ketones are reversible inhibitors of serine and cysteine proteases w7–9x, and they are inert towards enzymatic degradation. Therefore, we used them as ligands for the affinity purification of thermitase and cathepsins B, L, and S. Among other techniques, affinity chromatography has also been applied in the purification of antibodies w10x. The antigens, used as ligands, were often nonspecifically bound to the matrix, and this causes deformation of the antigen molecules and steric problems w11x. An oriented immobilization of the antigen on the support should be preferable. We present spacer-bound peptidyl chloromethyl ketones as tools for a specific and oriented immobilization of proteinases as antigens. Via the active site, immobilized cathepsin B was used to purify antibodies against human cathepsin B.
2. Experimental 2.1. Materials 2.1.1. Substrates Ala-4-nitroanilide hydrochloride ŽAla-pNA HCl., Benzoyl-Arg-2-naphthylamide ŽBzArg-bNA., Benzyloxycarbonyl-Phe-Arg-methylcoumarylamide ŽZ-Phe-Arg-MCA. and Z-Arg-Arg-MCA were purchased from Bachem ŽHeidelberg, Germany.. Pro-Ala-pNA. HCl was synthesized according to Ref. w7x, Z-Val-Val-Arg-MCA according to Ref. w12x and Succinyl-Ala-Ala-Phe-pNA ŽSuc-Ala-Ala-Phe-pNA. according to Ref. w13x. 2.1.2. Inhibitors Z-Ala-chloromethyl ketone ŽZ-AlaCK. and Z-Phe-AlaCK were synthesized as previously described by Ref. w8x. The peptidyl methyl ketones ŽMK. were prepared according to the procedures of Refs. w14,15x. 2.1.3. Supports Aminohexyl-Sepharose 4B ŽAH-Sepharose 4B. was obtained from Pharmacia ŽUppsala, Sweden. and Arginine-Agarose ŽC 12 -spacer. from Sigma ŽDeisenhofen, Germany.. Divicell, a cellulose-based support, activated with 5-norbornene-2,3-dicarboximido carbonochloridate Ž30–34 mmol of activated hydroxyl groups per ml sedimented gel., was a gift from Dr. H.-F. Boeden ŽCentral Institute of Molecular Biology, Berlin, Germany. ŽArzneimittelwerke, Leipzig, Germany.. and Dr. R. Muller ¨
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2.1.4. Enzymes AAP from Acinetobacter calcoaceticus was prepared according to Ref. w4x and MP-1 according to Ref. w5x. Subtilisins BPNX and Carlsberg were purchased from Boehringer ŽMannheim, Germany.. Subtilisin DY was a gift from Prof. N.C. Genov ŽBulgarian Academy of Sciences, Sofia, Bulgaria.. A concentrated supernatant of culture medium from Thermoactinomyces Õulgaris as well as pure thermitase, purified by isoelectric ŽHumboldt University, Berlin, Germany.. focusing, were a gift from Prof. W. Hohne ¨ Pure thermitase, purified by adsorption on porous glass beads, was a gift from Dr. U. Kettmann ŽMartin Luther University, Halle, Germany.. Cathepsin B from human liver and rat liver, respectively, were used as crude enzyme preparations after extraction, autolysis, acetone fractionation and DEAE-Cellulose ŽDiethylaminoethyl-Cellulose. chromatography according to Ref. w16x. 2.1.5. Antibodies The immunization scheme for goats was the same as described by Ref. w17x for rabbits. Immunoglobulins were prepared by the modified caprylic acid method w17x. 2.1.6. Other materials All chemicals were trade products with a high purity. The Diaflo ultrafiltration membranes YM-5, YM-10, and YM-30, used for the Amicon ultrafiltration systems, were purchased from Amicon ŽBeverly, MA, USA.. 2.2. Methods 2.2.1. Synthesis of AMK-sepharose 6X-N-ŽL-Alanine methyl ketone.-aminohexyl-Sepharose hydrochloride, AMK-Sepharose Ž5.: According to Scheme 1, Z-AlaCK Ž16 mg; 60 mmol., 2, was added to a suspension of AH-Sepharose 4B Ž1.5 g., 1, in 20 ml tetrahydrofuran ŽTHF.. N-Ethylmorpholine Ž10 ml; 79 mmol, NEM. was then added and the reaction mixture was stirred at 4 8C for 24 h. Product 3 was isolated by filtration, washed thoroughly with 100 ml THF and dried. Two milliliters HBrrglacial acetic acid Ž4 molrl. was added to the dry powder Ž1.5 g. of 3. The reaction was gently stirred at 4 8C. After 1 h, 30 ml diethyl ether was added, and the product 4 was separated by filtration. The solid was then washed with 50 ml ethylene glycol in water Ž10 vol.%. and, thereafter, with TrisrHCl buffer Ž0.05 molrl, pH 7.5. to yield compound 5. 2.2.2. Purification of the Acinetobacter alanyl aminopeptidase on AMK-sepharose A column Ž100 = 5 mm. of AMK-Sepharose Ž20–25 mmol AlaMK per g dry gel w4x. was equilibrated at 4 8C with TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100.. Then, 0.5 ml Triton X-100-containing membrane-protein extract Ž5.1 mg protein. w4x was applied to the column at a flow-rate of ca. 2 mlrh at 4 8C ŽFig. 1.. The fractionation was started after elution of ca. 3 ml. The column was then washed with 15 ml buffer. Gradient elution was carried out with increasing concentration of KCl Ž0–1.0 molrl. in
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Scheme 1. Synthesis of AMK-Sepharose as affinity chromatography matrix for purification of a membranebound AAP. 1: AH-Sepharose 4B, 2: Z-AlaCK, 5: AMK-Sepharose.
Fig. 1. Purification of the AAP from A. calcoaceticus by affinity chromatography on AMK-Sepharose. -'Absorbance at 280 nm Žprotein., -v- AAP activity ŽAla-pNA., -B- DPP IV activity ŽPro-Ala-pNA.; 0.5 ml Triton X-100-containing membrane protein extract was applied to the column Ž100=5 mm. at a flow-rate of ca. 2 mlrh at 4 8C. The fractionation was started after elution of ca. 3 ml. The gradient was carried out with increasing concentration of KCl in TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100..
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the TrisrHCl buffer. The AAP was eluted at 0.6 molrl KCl. The pooled fractions, containing pure Ala-pNA activity for AAP, were concentrated to ca. 4 ml Ž16 mgrml protein w18x., using an Amicon YM-30 membrane. Exopeptidase activities ŽFig. 1. were measured by incubation of 100-ml aliquotes of the eluent fractions with the chromogenic substrates Ž3 mmolrl. for 1 h at 37 8C and detection of the liberated 4-nitroaniline at l s 405 nm. Protein was assayed in the effluent at l s 280 nm. 2.2.3. Purification of the myeloma cell proteinase-1 on Arginine-Agarose A prepurified preparation of MP-1 Ž0.6 ml, 0.43 mg protein. w5x, after Sepharose 6B chromatography and concentration on an Amicon YM-30 membrane, was applied to a column Ž80 = 5 mm. of Arginine-Agarose ŽC 12 -spacer., equilibrated with TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100. at 4 8C. The flow-rate was 2 mlrh. The loaded column was then washed with 12 ml buffer for ca. 3 h. The bound MP-1 was eluted with increasing concentrations of NaCl Ž0–0.5 molrl. in the TrisrHCl buffer. Fractions Ž0.5 ml. were collected at a flow-rate of 2 mlrh. Protein was assayed in the effluent at l s 280 nm. MP-1 activity was detected by incubation of the eluent fractions Ž100 ml. with Bz-Arg-bNA Ž2 mmolrl. for 1 h at 37 8C ŽFig. 2a.. The enzymatically liberated 2-naphthylamine was measured at l s 340 nm. All fractions, containing Bz-Arg-bNA activity for MP-1, were pooled and concentrated to ca. 2.5 ml Ž38 mgrml protein w19x., using an Amicon YM-30 membrane. 2.2.4. Synthesis of DiÕicell-Aca-inhibitor affinity gels Activated Divicell gel was coupled with ´-aminocaproic acid ŽAca. in sodium tetraborate buffer Ž0.1 molrl, pH 8.0. to give support-spacer conjugates ŽDivicell-AcaOH. with 17–21 mmol carboxyl groups per ml sedimented gel. The N-deprotected peptidyl methyl ketones were coupled to the carboxyl groups of the spacer, using N-ethyl-N X-Ž3-dimethylamino-.propyl carbodiimide hydrochloride, at pH 4.5–5.0. Distilled water or, if the peptidyl derivative is insoluble in water, a dioxanrwater mixture was used as solvent. The gels were washed with 10 volumes of the gel volume with water, and then four times alternating with TrisrHCl buffer Ž0.1 molrl, pH 8.0, 0.5 molrl NaCl. and acetic acidracetate buffer Ž0.1 molrl, pH 4.0, 0.5 molrl NaCl.. All gels were stored in phosphate buffer Ž0.1 molrl, pH 6.0, 1.0 molrl NaCl, 0.02% NaN3 . at 4 8C. The affinity gels were characterized by amino acid analysis after total hydrolysis. The concentration of the immobilized ligands was determined to be between 12 and 17 mmolrml sedimented gel w15x. N-Deprotected peptidyl chloromethyl ketones were coupled to Divicell-Aca-OH using the same method. In the washing procedure, the time of exposure with TrisrHCl buffer Ž0.1 molrl, pH 7.5. was minimized. 2.2.5. Purification of thermitase on DiÕicell-Aca-Ala-Ala-PheMK A concentrated supernatant of culture medium of T. Õulgaris was prepurified by size-exclusion chromatography on Sephadex G-75 in ammonium acetate buffer Ž0.1 molrl, pH 7.5. at 4 8C. 65 ml Ž0.525 mg proteinrml. of the thermitase-containing fractions was applied to a column Ž6 = 50 mm. of Divicell-Aca-Ala-Ala-PheMK, equilibrated with the ammonium acetate buffer, at a flow-rate of 3 mlrh and at 4 8C.
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Fig. 2. Ža. Affinity chromatography of MP-1 from myeloma cells on Arginine-Agarose. -v- Absorbance 280 nm Žprotein., -B- MP-1 activity ŽBz-Arg-bNA.. A prepurified preparation of MP-1 was applied to the column Ž80=5 mm.. Subsequent elution of MP-1 was carried out with NaCl in TrisrHCl buffer Ž0.05 molrl, pH 7.5, 0.1% Triton X-100.. Žb. SDS polyacrylamide gel electrophoreses Ž12.5%, pH 8.8 w37x. of the purified MP-1 under reducing conditions with 0.1% SDS and 0.1% 2-mercaptoethanol, followed by Coomassie Blue staining. Lanes 2–5: purified MP-1, lanes 1 and 6: marker proteins ŽkDa.: 96, phosphorylase; 55, glutamate dehydrogenase; 39, aldolase; 30, carbonic anhydrase; 21, trypsin inhibitor.
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After washing with ammonium acetate buffer Žcontaining additionally 0.5 mmolrl CaCl 2 ., TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 . and, finally, addition of NaCl Ž1.0 mmolrl. to the TrisrHCl buffer, the protease was eluted with 2-propanol Ž40 vol.%., added to the TrisrHCl buffer, containing CaCl 2 and NaCl. During the washing procedure, the flow-rate was 12 mlrh. Elution was carried out at a flow-rate of 5 mlrh. The protein concentrations were determined by the Lowry et al. w18x method. 2.2.6. Purification of human cathepsin B on DiÕicell-Aca-Phe-PheMK A crude preparation of cathepsin B from human liver Ž5.5 ml, 1.06 mg proteinrml. in acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. was activated with dithioerythritol Ž10 mmolrl. and EDTA Ž10 mmolrl. and then slowly Žflow-rate, 3 mlrh. at 4 8C applied to a column Ž40 = 6 mm. of Divicell-Aca-Phe-PheMK. The affinity gel was washed with the same acetic acidracetate buffer, containing additionally NaCl Ž1.0 molrl., at a flow-rate of 5 mlrh. The bound enzyme was eluted with 40 vol.% ethylene glycol and 1 vol.% 2-mercaptoethanol in the washing buffer Žflow-rate, 4 mlrh.. All fractions, containing the same buffer, were pooled, and the pooled fractions were concentrated to ca. 2 ml, using an Amicon YM-5 membrane. The activity of cathepsin B was monitored with Z-Phe-Arg-MCA. In experiments with mixtures of cathepsins B and L, Z-Arg-Arg-MCA was additionally used and the enzymes discriminated as described in Ref. w16x. The enzymatically liberated methylcoumaryl amine was measured at lex s 380 nm, lem s 460 nm. The activity of cathepsin S in other experiments was monitored with Z-Val-Val-Arg-MCA. 2.2.7. Purification of cathepsin B-antibodies on an affinity matrix with oriented-immobilized cathepsin B 2.2.7.1. Immobilization of cathepsin B. A crude preparation of cathepsin B from rat liver was activated with dithioerythritol Ž10 mmolrl. in the presence of EDTA Ž10 mmolrl. and mixed with 10 ml affinity gel Divicell-Aca-Phe-AlaCK Žsediment, 3.5 ml. in acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. at room temperature. The suspension was shaken until the enzyme activity in the supernatant decreased to a negligible value. The gel was washed with distilled water and four times alternately with acetic acidracetate buffer Ž0.1 molrl, pH 5.0. and TrisrHCl buffer Ž0.1 molrl, pH 8.2., and then stored in the TrisrHCl buffer, containing additionally 0.02% NaN3 . 2.2.7.2. Preparation of the cathepsin B antibodies. An immunoglobulin fraction Ž60 mg proteinr2 ml. from goat antiserum against human cathepsin B in TrisrHCl buffer Ž0.02 molrl, pH 8.2. was slowly applied to a column Ž58 = 6 mm. of Divicell-Aca-Ala-Phecathepsin B at room temperature. After 30 min, the gel was washed with 10 ml of the TrisrHCl buffer and then with 10 ml of TrisrHCl buffer, containing additionally 1.0 molrl KCl. The antibodies to cathepsin B were eluted with 15 ml glycinerHCl buffer Ž0.2 molrl, pH 2.2. and collected in the same volume of TrisrHCl buffer Ž0.5 molrl, pH 8.5.. All fractions, containing the same buffer, were pooled and concentrated to ca. 1 ml, using an Amicon YM-10 membrane.
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3. Results and discussion 3.1. Purification of alanyl aminopeptidase from A. calcoaceticus The isolation of membrane-bound exopeptidases from a detergent-containing membrane protein solution by size-exclusion or ion-exchange chromatography is less successful. Affinity chromatography offers the possibility of enzyme-selective adsorption and subsequent recovery of the enzyme from an immobilized enzyme-specific ligand. The merits of immobilized inhibitors, dyes, or antibiotics for protease purification are explained by Ref. w1x. Active-site-directed reversible inhibitors of proteases are useful reagents in protein purification. Amino acid chloromethyl ketones and methyl ketones have been shown to be potent reversible inhibitors of soluble w9x and membrane-bound aminopeptidases w20x. Alanine methyl ketone is a strong competitive inhibitor Ž K i ( 10y6 molrl. w21x of Acinetobacter AAP. Alanine methyl ketone was chosen for immobilization because it binds to the aminopeptidase more strongly than the natural ligand alanine. Therefore, we synthesized alanine-methyl-ketone-modified AH-Sepharose ŽScheme 1. as affinity support. Alanine chloromethyl ketone was covalently bound to the AH-Sepharose by a linkage between the v-amino group of the alkyl side chain of the AH-Sepharose and the chloromethyl function of AlaCK w2x. This matrix was able to bind Acinetobacter AAP specifically and selectively ŽFig. 1.. The AAP was tightly adsorbed at pH 7.5 and 4 8C to the AMK-Sepharose matrix. Furthermore, Pro-Ala-pNA activity was bound weaker to the affinity adsorbent than the AAP. Non-adsorbed proteins were washed out with TrisrHCl buffer at pH 7.5. The specific complex of the AAP with the immobilized alanine methyl ketone could be decomposed with KCl. With an increasing KCl gradient, the Pro-Ala-pNA activity ŽDPP IV. was eluted as first peak at ca. 0.20 molrl KCl ŽFig. 1.. The AAP peak was eluted at 0.6 molrl KCl in good yield Ž78% of applied Ala-pNA activity.. It is suggested that the AAP, a SH-dependent exopeptidase of 212 kDa w4x, forms a thiohemiacetal in the active site of AAP with the carbonyl function of the immobilized alanine methyl ketone ligand at the affinity matrix. The mechanism is likely to be the same as that shown for the inhibition of AAP by soluble alanine methyl ketone w20x. Endopeptidases were not bound by AMK-Sepharose. The gel was stable and the AMK-ligand was not hydrolyzed by enzymes. The homogeneity of the AAP from A. calcoaceticus was confirmed by polyacrylamide gel electrophoresis w4x. From these results, it is evident that affinity chromatography is a very useful method for the purification of detergent solubilized exopeptidases. 3.2. Purification of myeloma cell proteinase The usual procedures of protein separation and purification are based on the relatively small differences in shape, size, and charge of water-soluble proteins in the mixture. In contrast, detergent-solubilized membrane-bound proteinases exist in a detergent-protein mixed micelle form. A good approach to the purification of micellanous associated membrane proteases employs affinity chromatography on proteinase-selective ligands.
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The MP-1 is a membrane-bound proteinase from the plasma membrane of myeloma cells. The enzyme was identified as a trypsin-like peptidase. MP-1 cleaves the substrate Bz-Arg-bNA optimally at pH 7.5 at 37 8C w6x. After treatment of the plasma membrane fragments with Triton X-100, a soluble membrane protein mixture, containing MP-1, was obtained. It was not possible to purify MP-1 from this protein mixture by size-exclusion or ion-exchange chromatography. MP-1 was contaminated with membrane-type matrix metalloproteinases w22x, cathepsin, and serine protease activities w23x. Arginine-Agarose with C 12 -spacer chains was shown to be an effective substrate-like affinity sorbent for MP-1 in contrast to Arginine-Agarose without spacer, which did not adsorb MP-1. Fig. 2a represents the affinity chromatography of the MP-1 from a Triton X-100-containing membrane protein extract, pretreated on a Sepharose 6B column. The contaminated MP-1 solution was applied to the affinity column. After washing off non-adsorbed membrane proteins with TrisrHCl buffer, the MP-1 was eluted with a linear gradient of NaCl in a yield of 89% of the applied activity and in very high purity. The homogeneity of MP-1 was demonstrated by polyacrylamide gel electrophoresis ŽFig. 2b. and Western blot analysis. MP-1 was found to consist of four identical subunits with molecular weights of 64 kDa ŽSDS-PAGE.. Arginine-specific proteinases, like cathepsins or plasminogen activator, were detectable only in the washing buffer eluate but not in the gradient fractions. The desorption of MP-1 was also successful in the presence of Triton X-100 concentrations of 1% in the buffer. 3.3. Affinity purification of thermitase and related proteases Peptidyl methyl ketones as ligands of proteinases are effective competitive inhibitors for some subtilisins and related proteinases, especially thermitase w14,24,25x, and, therefore, suitable as ligands in affinity chromatography. According to the specificity of the subtilisins and subtilisin-related enzymes, the most efficient peptidyl methyl ketone was found to be Z-Ala-Ala-PheMK with an inhibition constant Ž K i . of 3.0 = 10y7 molrl for thermitase, 9.4 = 10y6 molrl for subtilisin Carlsberg, 3.1 = 10y5 molrl for subtilisin DY, 1.3 = 10y5 molrl for proteinase K, and 1.3 = 10y4 molrl for subtilisin BPNX , whereas a-chymotrypsin was inhibited only with a K i of 1.1 = 10y3 molrl w13,25x. It has been shown that the subtilisins Carlsberg and DY as well as proteinase K and thermitase are quantitatively adsorbed on affinity gels with Aca as spacer and Ala 2-PheMK as ligand w3,15x. As support, Divicell is preferable to Sepharose since Divicell has advantages with respect to the binding capacity and to the stability of the gel, which causes to the very stable urethane bonding between support and spacer w26,27x. The elution pattern of a Sephadex G-75-prepurified thermitase preparation from Divicell-Aca-Ala-Ala-PheMK is shown in Fig. 3. The adsorbed thermitase was eluted in a sharp peak by of 2-propanol Ž40 vol.%.. 2-Propanol reduces dramatically the enzymatic activity. This effect, however, is completely reversible and has the advantage of suppressing autolysis, which otherwise destroy considerable amounts of the active enzyme w15x. The specific activity was increased from 1.3 to 3.0 mkatrmg protein by the affinity chromatography. Fig. 4 shows the electrophoresis pattern of the Sephadex G-75-prepurified thermitase and of the purified thermitase after affinity chromatography in comparison with preparations, purified by two other methods w28,29x. Divicell-Aca-
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Fig. 3. Affinity chromatography of thermitase on Divicell-Aca-Ala-Ala-PheMK. -v- Absorbance at 280 nm Žprotein., -B- relative activity against Suc-Ala-Ala-Phe-pNA: activity of the eluateractivity of the applied solution. Dotted lines indicate the change of the eluent. 1: applied solution—65 ml Sephadex G-75-prepurified thermitase Žprotein: 0.525 mgrml. in ammonium acetate buffer Ž0.1 molrl, pH 7.5.; 2: ammonium acetate buffer Ž0.1 molrl, pH 7.5, 0.5 mmolrl CaCl 2 .; 3: TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 .; 4: TrisrHCl buffer Ž0.1 molrl, pH 7.8, 0.5 mmolrl CaCl 2 , 1.0 molrl NaCl.; 5: TrisrHCl buffer Ž0.1 molrl; pH 7.8, 0.5 mmolrl CaCl 2 , 1.0 molrl NaCl, 40 vol.% 2-propanol.. Column 50=6 mm; flow-rates, 1: 3 mlrh; 2–4: 12 mlrh; 5: 5 mlrh.
Ala-Ala-PheMK proved very suitable for purification of thermitase, although the immobilized inhibitor Ž K i ca. 3.5 = 10y6 molrl. was ca. 100 times less potent than Z-Ala-Ala-PheMK and ca. 10 times less potent than Ac-Aca-Ala-Ala-PheMK, mimicking the spacer-ligand unit w15x. Subtilisins and proteinase K have been eluated from Divicell-Aca-Ala-Ala-PheMK with lower concentrations of 2-propanol than thermitase according to their decreased inhibition by Z-Ala-Ala-PheMK. 3.4. Affinity purification of the cathepsins B, L and S Since peptidyl methyl ketones are inhibitors not only of a number of serine proteases, but also of cysteine proteases Žpapain, cathepsins B, L, and S w12,30,31x., we tried to find useful affinity matrices with peptidyl methyl ketones as ligands and favorable chromatographical conditions for the purification of the cathepsins mentioned. DivicellAca-Phe-AlaMK and Divicell-Aca-Phe-PheMK were tested as affinity media for the purification of cathepsins B and L. Only Divicell-Aca-Phe-PheMK proved a suitable
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Fig. 4. SDS polyacrylamide gel electrophoresis of thermitase. Conditions: 0.1% SDS, 15% polyacrylamide gel ŽpH 8.8., Coomassie Blue staining. In order to prevent autolysis during the boiling with SDS, thermitase was denaturated with 20% trichloroacetic acid ŽLanes 1 and 2. and incubated with Z-Ala-Ala-PheCK for 90 min. ŽLanes 3–5., respectively, before treatment with SDS. Lanes: 1, crude product; 2, prepurified thermitase after size-exclusion chromatography with Sephadex G-75; 3, after affinity chromatography; 4, after isoelectric focusing; 5, after adsorption on porous glass beads; 6, marker proteins ŽkDa.: 96, phosphorylase; 66, albumin; 45, ovalbumin; 30, carbonic anhydrase; 21, trypsin inhibitor; 14.4, a-lactalbumin.
affinity gel for purifying the enzymes. A similar elution profile of cathepsin B, as shown in Fig. 5, was obtained with cathepsin L. Buffer 3 eluted 62% of the applied activity of cathepsin B and 11% of the applied protein ŽFig. 5.. The total recovery was 69% of the protein and 85% of the activity. Western blot analysis with polyclonal antibodies to human cathepsin B and human cystatins A and B revealed the presence of different concentrations of cathepsin B in all fractions, shown in Fig. 5, and, in addition, complexes of cathepsin B with the cystatins. Peptidyl diazomethyl ketones have often been described as selective irreversible inhibitors of cysteine proteases w32x, but they also inhibit some serine proteases w33x. The inhibitor, which is most discriminating between cathepsin B and cathepsin L, is Z-Phe-TyrŽO-t-Bu.CHN2 w34x. The inactivation rate k inactrK i for cathepsin L is 1.2 = 10 7 My1 miny1 , for cathepsin B, 6.2 = 10 2 My1 miny1 , leading to a relative inactivation rate LrB of ca. 20.000 w34x. These kinetic data suggested that the dipeptidyl methyl ketone Phe-TyrŽO-t-Bu.MK could be a suitable affinity ligand for separating cathepsins B and L from a mixture of the enzymes. However, use of Divicell-Aca-Phe-TyrŽO-tBu.MK for the separation of cathepsin B and L did not come up to our expectations. Only an enrichment of each enzyme, but not a complete separation, could be achieved. Ca. 75% of the applied cathepsin B activity was eluted without retention, whereas cathepsin L was almost completely bound to the affinity gel and eluted by additions of NaCl, ethylene glycol, and 2-mercaptoethanol to the acetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA. used.
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Fig. 5. Affinity chromatography of human cathepsin B on Divicell-Aca-Phe-PheMK. -v- Absorbance 278 nm Žprotein., -B- activity against Z-Phe-Arg-MCA. Dotted lines indicate the change of the eluent. 1: applied solution—5.5 ml crude cathepsin B Žprotein: 1.06 mgrml. in standard acetic acidracetate buffer Ž0.1 molrl, pH 5.5, 1.0 mmolrl EDTA.; 2: standard buffer, containing NaCl Ž1.0 molrl.; 3: standard buffer, containing NaCl Ž1.0 molrl., ethylene glycol Ž40 vol.%., and 2-mercaptoethanol Ž1 vol.%.; 4: the affinity gel was left overnight in buffer 3. Column: 40=6 mm; flow-rates, 1: 3 mlrh; 2, 3: 5 mlrh, 4: 4 mlrh.
Inhibition of cathepsin S by Z-Val-Val-PheMK has a K i value of 1.8 = 10y6 molrl w12x. Therefore, we tested Divicell-Aca-Val-Val-PheMK as affinity support for cathepsin S from bovine spleen. Cathepsin S bound completely at pH 7.0 Ž0.1 molrl phosphate buffer. to the gel. After washing to remove contaminating proteins, the enzyme was eluted at pH 5.0 Ž0.1 molrl acetic acidracetate buffer. with additions of NaCl, 2-propanol and 2-mercaptoethanol to the buffer. 3.5. Purification of anti-cathepsin B on affinity-immobilized cathepsin B In the affinity purification of antibodies, affinity supports have been used mainly where the antigen molecules are nonspecifically bound to the spacer arms or directly to the matrix via amino or carboxyl groups at the surface of the antigen w11x. The consequence of such a coupling is that each of the antigen molecules is nonspecifically and in a different manner immobilized. It is assumed that a large number of antigen molecules are linked to the support via several functional groups, causing deformation of the molecules. Problems with respect to the steric accessibility of the antigen may also
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occur w11x. Therefore, oriented immobilization of proteases, acting as antigens, has the following advantages: Ža. all antigen molecules are immobilized in the same manner via one center, Žb. no significant deformation occurs, Žc. all molecules present the same epitopes to the antibodies, Žd. the affinant often shows an increased stability. We propose to use specific spacer-bound peptidyl chloromethyl ketones for the covalent immobilization of proteases via their active site since chloromethyl ketones react spontaneously with cystein proteases by S-alkylation w35x and with serine proteases by N-alkylation w36x, respectively, of their catalytic sites. The formed modified matrix, consisting of support, spacer, peptide chain and protease Žantigen., is an affinity sorbent for antibodies to the immobilized protease. We synthesized Divicell-Aca-Phe-AlaCK for the irreversible binding of cathepsin B. The immunoglobulin fraction of an antiserum from goat against human cathepsin B was applied in TrisrHCl buffer ŽpH 8.2. to an affinity column of Divicell-Aca-Phe-AlaCH 2-cathepsin B. Ca. 42% and 11%, respectively, of the applied protein was washed out in two steps. The antibodies to cathepsin B Žca. 15%. were eluted at pH 2.2 with a glycinerHCl buffer, as revealed by Western blots with cathepsin B. Anti-cathepsin B could not be detected in any other fraction. This method has the advantage that crude preparations may serve as a source of proteases to be immobilized because of selective binding of the enzyme. It is to assume that most of the epitopes at the surface of the antigen-protease are not deformed, and, thus, the antibodies could bind in an optimal manner and could be obtained in maximal yield.
References w1x Ibrahim-Granet O, Bertrand O. Separation of proteases: old and new approaches. J Chromatogr, B: Biomed Sci Appl 1996;684:239–63. w2x Jahreis G, Aurich H. Synthese einer Alaninmethylketon-modifizierten AH-Sepharose zur affinitats¨ chromatographischen Reinigung einer bakteriellen Alaninaminopeptidase. Pharmazie 1987;42:270. w3x Peters K, Fittkau S. Affinitatschromatographie von Proteasen mit Peptidmethylketonen als Liganden. ¨ Biomed Biochim Acta 1990;49:173–8. w4x Jahreis G, Sorger H, Aurich H. Eine membrangebundene Alaninaminopeptidase aus Acinetobacter calcoaceticus: 1. Isolierung und Reinigung des Enzyms. Biomed Biochim Acta 1989;48:617–24. w5x Jahreis G, Preissner A, Weber E. A new growth releated endopeptidase from myeloma cells. Biol Chem Hoppe-Seyler 1994;375:58. w6x Jahreis G, Weber E, Lutz D, Preissner A. Characterization of a new endopeptidase from myeloma cells. Biol Chem Hoppe-Seyler 1995;376:S111. w7x Heins J, Welker P, Schonlein C, Born I, Hartrodt B, Neubert K, et al. Mechanism of proline-specific ¨ proteinases: substrate specificity of dipeptidyl peptidase IV from pig kidney and proline-specific endopeptidase from FlaÕobacterium meningosepticum. Biochim Biophys Acta 1988;954:161–9. w8x Jahreis G, Smalla K, Fittkau S. Thermitase—eine thermostabile Serinprotease: 2. Synthese von substratanalogen Peptidchlormethylketonen als potentielle irreversible Enzyminhibitoren. J Prakt Chem 1984;326:41–7. w9x Kettner C, Glover GI, Prescott JM. Kinetics of inhibition of Aeromonas aminopeptidase by leucine methyl ketone derivatives. Arch Biochem Biophys 1974;165:739–43. w10x Nau DR. Chromatographic methods for antibody purification and analysis. BioChromatography 1989; 4:4–17. w11x Turkova J. Oriented immobilization of biologically active proteins as tool for revealing protein interactions and function. J Chromatogr 1999;722:11–31.
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