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`Page 1 ofll
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`CSL V. Shire
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`CSL EXHIBIT 1042
`CSL v. Shire
`
`

`

`2A
`
`B I 0 C H E M I S T R Y , V 0 L . 2 2 , N 0 . 2 I ,
`
`I 9 8 3
`
`HANS NEURA TH, Department of Biochemistry
`University of Washington, Seattle, Washington 98195
`(206) 543-1690
`
`EDITOR
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`ASSOCIATE EDITORS
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`Page 2 of 11
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`

`

`Biochemistry®
`
`BICHAw 22(21) 4867-5054 (1983)
`ISSN 0006-2960
`
`a biweekly publication of the American Chemical Society
`
`Registered in U.S. Parent and Trademark Office; Copyright 1983 by the American Chemical Society
`
`October 11, 1983
`
`ACCELERATED PUBLICATIONS
`
`4867 Epidermal Filaggrin Is Synthesized on a Large Messenger Ribonucleic Acid as a
`High-Molecular-Weight Precursor
`Rick L. Meek, John D. Lonsdale-Eccles, and Beverly A. Dale*
`
`ARTICLES
`
`4872 Deoxyribonucleic Acid Damage by Neocarzinostatin Chromophore: Strand Breaks Generated by Selective
`Oxidation of C-5' of Deoxyribose
`Lizzy S. Kappen and Irving H. Goldberg*
`
`4878 Acridine-Psoralen Amines and Their Interaction with Deoxyribonucleic Acid
`John Bondo Hansen, Tor ben Koch, Ole Buchardt, * Peter E. Nielsen, Michael Wirth, and
`Bengt Norden
`
`4887 Periodicity of Exonuclease III Digestion of Chromatin and the Pitch of Deoxyribonucleic Acid on the Nucleosome
`Ariel Prunell
`
`4894 Secondary Structure Assignment for a/(3 Proteins by a Combinatorial Approach
`Fred E. Cohen, Robert M. Abarbanel, I. D. Kuntz,* and Robert J. Fletterick
`
`4905
`
`Influence of Local Nucleotide Sequence on Substitution of 2-Aminopurine for Adenine during Deoxyribonucleic
`Acid Synthesis in Vitro
`Reynaldo C. Pless and Maurice J. Bessman*
`
`4916 Neutron Scattering Studies of Nucleosome Structure at Low Ionic Strength
`Edward C. Uberbacher, Venkatraman Ramakrishnan, Donald E. Olins, and Gerard J. Bunick*
`
`4'J24 Circular Dichroism and Ordered Structure of Bisnucleoside Oligophosphates and Their Zn2+
`and Mg2+ Complexes
`Eggehard Holler, Barton Holmquist, Bert L. Vallee, Krishan Taneja, and Paul Zamecnik*
`
`4933 Reaction of Human arMacroglobulin Half-Molecules with Plasmin as a Probe of Protease
`Binding Site Structure
`Steven L. Gonias and Salvatore V. Pizzo*
`
`4940
`
`Isolation and Characterization of Pepsin-Solubilized Human Basement Membrane (Type IV) Collagen Peptides
`RobertS. Mac Wright,* Virginia A. Benson, Katherine T. Lovello, Michel van der Rest, and
`Peter P. Fietzek
`
`4949 Photoinactivation of Agaricus bisporus Tyrosinase: Modification of the Binuclear Copper Site
`David C. Fry and Kenneth G. Strothkamp*
`
`4954 Role of Magnesium Binding to Myosin in Controlling the State of Cross-Bridges in Skeletal Rabbit Muscle
`Emil Reisler,* Jane Liu, and Pearl Cheung
`
`496! Reinvestigation of the Shape and State of Hydration of the Skeletal Myosin Subfragment 1 Monomer in Solution
`Manuel Garrigos, Jean E. Morel,* and Jose Garcia de Ia Torre
`
`4969
`
`Formation of a Supramolecular Complex Is Involved in the Reconstitution of Basement Membrane Components
`Hynda K. Kleinman,* Mary L. McGarvey, John R. Hassell, and George R. Martin
`
`Page 3 of 11
`
`

`

`---
`
`BIOCHEMISTRY, VOL. 22, NO. 21, 1983
`
`5A
`
`4974 Natural Abundance Carbon-13 Nuclear Magnetic Resonance Spectroscopy of Liver and Adipose Tissue
`of the Living Rat
`Paul Canioni, * Jeffry R. Alger, and Robert G. Shulman
`
`4980 Chemical Structure and Antigenic Aspects of Complexes Obtained from Epimastigotes of Trypanosoma cruzi
`Lucia Mendons:a-Previato, Philip A. J. Gorin,* Arnaldo F. Braga, Julio Scharfstein, and
`Jose 0. Previato
`
`4987
`
`Functions of the 5'-Phosphoryl Group of Pyridoxal 5'-Phosphate in Phosphorylase: A Study Using
`Pyridoxal-Reconstituted Enzyme as a Model System
`Yen Chung Chang, Timothy McCalmont, and Donald J. Graves*
`
`4994 Effect of Oxyanions of the Early Transition Metals on Rabbit Skeletal Muscle Phosphorylase
`Gopalan Soman, Yen Chung Chang, and Donald J. Graves*
`
`5001 Human CI Inhibitor: Improved Isolation and Preliminary Structural Characterization
`Richard A. Harrison
`
`5008 High Mobility Group Proteins: Abundance, Turnover, and Relationship to Transcriptionally Active Chromatin
`Ronald L. Seale,* Anthony T. Annunziato, and Richard D. Smith
`
`5015
`•
`
`Investigation of the Structure, Protonation, and Reactivity of Tetraammine(imidodiphosphato)cobalt{III), a
`Substrate for Potato Apyrase
`TuliP. Haromy, Wilson B. Knight, Debra Dunaway-Mariano,* and M. Sundaralingam*
`
`5022 Conversion of Nerve Growth Factor-Receptor Complexes to a Slowly Dissociating, Triton X-100 Insoluble State
`by Anti Nerve Growth Factor Antibodies
`Ronald D. Vale and Eric M. Shooter*
`
`5029 Subcellular Distribution of Cholic Acid:Coenzyme A Ligase and Deoxycholic Acid:Coenzyme A Ligase
`Activities in Rat Liver
`F. Anthony Simion, Becca Fleischer,* and Sidney Fleischer
`
`5034 Glycolipids of Fetal, Newborn, and Adult Erythrocytes: Glycolipid Pattern and Structural Study of
`HrGlycolipid from Newborn Erythrocytes
`Michiko N. Fukuda* and Steven B. Lcvery
`
`5041
`
`Incorporation of Ganglioside Analogues into Fibroblast Cell Membranes. A Spin-Label Study
`Gunter Schwarzmann, * Peter Hoffmann-Bleihauer, Jorg Schubert, Konrad Sandhoff, and
`Derek Marsh
`
`5048 Electron Paramagnetic Resonance and Optical Spectroscopic Evidence for Interaction between Siroheme and
`Tetranuclear Iron-Sulfur Center Prosthetic Groups in Spinach Ferredoxin-Nitrite Reductase
`James 0. Wilkerson, Peter A. Janick, and Lewis M. Siegel*
`
`• Supplementary material for this paper is available separately (consult the masthead
`page for ordering information); it will also appear following the paper in the microfilm
`edition of this journal.
`
`* In papers with more than one author, the asterisk indicates the name of the author to
`whom inquiries about the paper should be addressed.
`
`Page 4 of 11
`
`

`

`Biochemistry 1983, 22, 5001-5007
`
`5001
`
`J-luman Cl Inhibitor: Improved Isolation and Preliminary Structural
`Characterization t
`
`Richard A. Harrison*
`
`ABSTRACT: ~n improved procedure for the isolation of the CI
`inhibitor (C1-INH) component of human complement is re(cid:173)
`ported. Following preliminary steps to remove plasminogen,
`fib ri nogen, and aggregated material, three conventional
`cltromatographic steps a re u~ed to isolate Ci-INH in high
`(70%) overall yield. An extinction coefficient (Ei~~~01) of 3.60
`has been determined. T\\e isolated protein exhibits a single
`band on sodium dodecyl sulfate-polyacrylamide gel electro(cid:173)
`phoresis, with a mobility corresponding to an apparent mo(cid:173)
`lecular weight (M,) of 105 000. After removal of carbohy(cid:173)
`drate, the protein shows an increased mobility, corresponding
`to an apparent M, of 78 000. A total earbohydrate content
`of 33% has been calculated, and from this and the size of the
`deglycosylated polypeptide, a true molecular weight of 116 000
`was estimated. Further analysis of the carbohydrate has in(cid:173)
`dicated a galactose:mannose ratio of 2:1 and approximately
`equimolar amounts of N-acetylglucosamine and N-acetyl-
`
`Cr inhibitor (CI-INH) 1 is the major homeostatic component
`
`of the classical pathway of complement activation, inhibiting
`the proteolytic activity of activated C1 and thus the activation
`of C4 and C2. It was first described as a serum a 2-globulin
`with inhibitory activity against CI esterase (Ratnoff & Lepow,
`1957; Pensky eta!., 1961) and later shown to be identical with
`the a 2-neuraminoglycoprotein described by Schultze and co(cid:173)
`workers (Schultze eta!., 1962; Pensky & Schwick, 1969).
`Analysis of the mechanism by which CI-INH inhibits CI has
`shown it to act in a stoichiometric fashion with both Cir and
`Ci s, forming 1:1 molar complexes (Harpel & Cooper, 1975;
`Sim eta!., 1979b, 1980). The interaction between CI-INH
`and both Cir and Cis is covalent and believed to involve the
`active-site serine of the proteases (Arlaud et al., 1979). In
`this respect, it appears to have a reaction mechanism analogous
`to those of other plasma protease inhibitors such as a 2-anti(cid:173)
`plasmin, anti-thrombin III, and a 1-protease inhibitor (Cohen,
`1973; Moroi eta!., 1975; Owen, 1975; Baugh & Travis, 1976;
`Moroi & Aoki, 1977; Wiman & Collen, 1979). In addition
`to this direct inhibitory role, it has recently been shown that
`CI-INH promotes dissociation of CI bound to immune com(cid:173)
`plexes, releasing a Cir-Cis-(CI-INHh complex (Sim eta!.,
`1979a; Ziccardi & Cooper, 1979).
`Little is known about the structure of CI-INH. It is heavily
`glycosylated, with a reported carbohydrate content of 35%
`(Haupt et a!., 1970), and consists of a single polypeptide chain.
`Its molecular weight has been estimated as about 105 000 by
`both NaDodSOcpolyacrylamide gel electrophoresis and
`analytical ultracentrifugation (Pensky et a!., 1961; Haupt et
`
`galactosamine. This composition is unusual for a plasma
`protein and suggests that much of the carbohydrate is con(cid:173)
`tained in linkages other than the typical N-glycosidic struc(cid:173)
`tures. Values found for the amino acid composition are
`compared with those reported previously. The amino-terminal
`sequence ( 40 residues) of CI-INH is also reported. Asparagine
`lies at the amino terminus. Neither high-performance liquid
`chromatography of the released phenylthiohydantoin derivative
`nor back-hydrolysis of the thiazolinone permitted identification
`of the residue contained at position 3. The sequence around
`this position is compatible, however, with an N-glycosidic
`linkage to residue 3. The first 10 residues also contain an
`unusual run of 5 hydroxyl-containing amino acids (-Thr(cid:173)
`Ser-Ser-Ser-Ser-) at positions 5-9. Visual comparison of the
`amino-terminal seq1.1ences with those reported for other pro(cid:173)
`tease inhibitors does not indicate any sequence homology.
`
`a!., 1970; Harpel & Cooper, 1975; Reboul et a!., 1977).
`Recent electron micrographic analysis has suggested that
`CT-INH is a highly elongated molecule containing rodlike and
`globular domains (Odermatt eta!., 1981). This fits well with
`its low sedimentation coefficient of 3.7-4.5 S. Amino acid and
`carbohydrate compositions of the protein have also been
`published (Haupt eta!., 1970; Harpel eta!., 1975).
`While CT-INH was first described as an inhibitor of acti(cid:173)
`vated C1, it has long been known that it is active against
`several other serum proteases. These include components of
`the coagulation system (factors Xla and Xlla; Forbes et a!.,
`1970), the fibrinolytic system (plasmin; Ratnoff eta!., 1969),
`and the kinin system (kallikrein; Gigli eta!., 1970). However,
`CI-INH is the only known plasma inhibitor of Ci r and Cs.
`Other plasma proteases that can be inhibited by CI-INH have
`alternative regulatory pathways. For example, plasmin is
`inhibited by a 2-antiplasmin (Moroi & Aoki, 1976; Collen,
`1976; Mullertz & Clemmensen, 1976), and kallikrein can be
`inhibited by armacroglobulin (Schapira eta!., 1981, 1982).
`It seems probable, therefore, that its major physiological role
`is directed against complement activation via the classical
`pathway. These secondary activities, particularly that against
`kallikrein, remain of interest because of the disease hereditary
`angioneurotic edema (HANE), which is associated either with
`low levels of apparently normal CI-INH (Donaldson & Evans,
`1963) or with the presence of a dysfunctional CI-INH protein
`(Rosen eta!., 1965, 1971).
`The work reported here was initiated in order that the in(cid:173)
`teractions between both the normal and dysfunctional CI-INH
`
`1 From the Division of Immunology, Children s Hospital Medical
`Center and Department of Pediatrics, Hs!Vard Medical School, Boston,
`Massachu~etts 02115. Receiued February 18, /983. Supported by grants
`from the Birth Defects Fou.ndation- March of Dimes and Grants A I
`05877 and AI 15033 of the U.S. Public Health Service.
`*Address correspondence to this author at the MRC Unit on Mech(cid:173)
`anisms in Tumour Immunity, MRC Centre, Cambridge CB2 2QH,
`England.
`.
`
`1 Abbreviations: CI-INH, CI esterase inhibitor [all other complement
`nomenclature follows the recommendations of the W.H.O. Committee
`on Complement (1968)]; HANE, hereditary angioneurotic edema; PEG,
`poly(ethylene glycol); NaDodS04, sodium dodecyl sulfate; TFMSA,
`trifluoromethanesulfonic acid; PTH, phenylthiohydantoin; Tris, tris(hy(cid:173)
`droxymethyl)aminomethane; EDT A, ethylenediaminetetraacetic acid;
`PAS, periodic acid-Schiff; HPLC, high-performance liquid chromatog(cid:173)
`raphy.
`
`0006-2960/83/0422-5001$01.50/0
`
`© 1983 American Chemical Society
`
`Page 5 of 11
`
`

`

`5002 BIOCHEMISTRY
`
`proteins and Cis or other plasma proteases, particularly
`kallikrein, could be investigated. Parallel chemical charac(cid:173)
`terization of the normal and dysfunctional proteins would also
`permit definition of the structural lesions leading to CI-INH
`dysfunction. Plasma containing dysfunctional proteins, though
`fresh frozen, were frequently several years old (and no longer
`obtainable). In addition, only limited volumes were available.
`Considerable attention was therefore spent in developing an
`isolation procedure, applicable to both normal and dysfunc(cid:173)
`tional proteins, with special emphasis on improving the yield
`of CI-INH from long-term-stored plasmas. This, together
`with preliminary chemical characterization of the isolated
`functional protein, is reported here.
`
`Materials and Methods
`DEAE-Sephadex A 50, Sephadex G 150 superfine, and Se(cid:173)
`pharose CL6B were purchased from Pharmacia. Lysine(cid:173)
`Sepharose was synthesized according to Deutsch & Mertz
`(1970). Hydroxylapatite (Bio-Gel HTP) was from Bio-Rad,
`as were NaDodS04, acrylamide, methylenebis(acrylamide),
`N,N,N',N'-tetramethylethylenediamine, Coomassie brilliant
`blue R250, and Basic Fuchsin. Dithiothreitol was from
`Calbiochem and iodoacetic acid from Kodak. Iodo[1- 14C](cid:173)
`acetic acid was from New England Nuclear. All sequenator
`reagents and solvents as well as methanol, norleucine, and
`PTH-norleucine were purchased from Pierce. Acetyl chloride
`was purchased from Sequemat, and acetonitrile and di(cid:173)
`chloroethane were from Burdick and Jackson. Anhydrous
`TFMSA was supplied by Sigma and anisole by Aldrich.
`Monospecific goat anti-human CI-INH was provided by
`Atlantic Antibodies. Fresh frozen plasma was obtained from
`the American Red Cross Blood Services, Northeast Region.
`Occasionally, freshly drawn plasma from fasted volunteers was
`used.
`CI-INH was located in column effluents by single-dimen(cid:173)
`sion crossed immunoelectrophoresis (Laurell, 1966) and
`quantitated antigenically by double diffusion in agar (Mancini
`et al., 1965). Functional assays were performed by using the
`procedure of Gigli and co-workers (Gigli et al., 1968). Protein
`solutions were concentrated by ultrafiltration using PM10
`membranes (Amicon). Phosphate buffers were made by using
`KH 2P04 and adjusting the pH with NaOH. NaDodSOc
`polyacrylamide gel electrophoresis was performed according
`to Laemmli (1970). Samples were incubated at 100 °C for
`2 min in sample buffer containing 0.1 M 2-mercaptoethanol
`prior to loading and the gels stained either with Coomassie
`brilliant blue R250 or by the periodic acid-Schiff (PAS)
`procedure (Segrest & Jackson, 1972).
`Carbohydrate was removed from CI-INH by incubation
`in anhydrous TFMSA (Edge et al., 1981). Briefly, the pro(cid:173)
`cedure used was as follows: 0.5 mL of anhydrous TFMSA
`containing 5% anisole was added to 10 mg of salt-free lyo(cid:173)
`philized protein in a conical stoppered glass tube; the tube was
`flushed with nitrogen, stoppered, and sealed with Parafilm.
`The dissolved protein was incubated at 0 °C for 3 h (during
`which time a faint pint color developed) and then the reaction
`terminated by the gradual addition, with vortexing, of an(cid:173)
`hydrous sodium carbonate to neutralize the acid. The neu(cid:173)
`tralized reaction wa tra nsferred with excess water to a djalysis
`bag (Spectrapor 2); excess salt, reactants, and released sugars
`were removed by dialyt>is against distilled water, and the
`precipitated polypeptide was recovered by lyophilization.
`Alkylation was performed by using the following procedure.
`Lyophil ized proteins (I 0 mg) was dissolved in 1 mL of 0.2 M
`Tris-HCI/6.0 M guanidine, pH 8.0. Dithiothreitol was then
`added to 5 mM, and the reaction vessel was flushed with
`
`HARRISON
`
`nitrogen, sealed, and incubated at 25 o'C for 30 min. The
`reduced protein was then radioalkylated by the addition of
`iodo[1- 14C]acetic acid (adjusted to a specific activity of about
`10 mCi/mmol) at a final concentration of 20 mM. There(cid:173)
`action was carried out (25 °C, 60 min) under nitrogen in the
`dark and terminated by the addition of 0.1 mL of 2-
`mercaptoethanol. Excess reactants were removed by dialysis
`against water, and the protein was recovered by lyophilization.
`Samples for amino acid hydrolysis were hydrolyzed (24-96
`h;.110°C) under vacuum in 6.0 M HCl (Ultrex) containing
`1% phenol and analyzed with a Beckman 121MB analyzer.
`Samples were prepared for carbohydrate analysis by hydrolysis
`for 24 h in 1 M methanolic HCl at 85 °C. Mannitol was used
`as an internal standard. Trimethylsilylation of the liberated
`sugars [see Bhaskar & Reid (1981)] was performed prior to
`analysis by gas-liquid chromatography (Clamp et al., 1972).
`Automated Edman degradation was performed by using a
`Beckman 890C sequencer modified with a cold trap. Con(cid:173)
`version was performed either in 1 M HCl containing 1.0%
`ethanethiol (Hermondson et al., 1972) or, in those sequencer
`runs for which the instrument was equipped with a P-6 au(cid:173)
`toconverter (Sequemat), in methanolic HCl (10 min, 65 °C;
`acetyl chloride:methanol1:7 by volume). A 0.1 M Quadrol
`program (Brauer et al., 1975) was used, and two coupling
`cycles were performed before the initial cleavage reaction.
`Phenylthiohydantoin derivatives were identified by high-per(cid:173)
`formance liquid chromatography. A Zorbax ODS column (Du
`Pont Instruments) equilibrated in 0.01 M sodium acetate, pH
`5.5, and developed with acetonitrile gradients modified from
`those of Zalut and co-workers (Zalut et al., 1980) was used.
`The identification of certain residues was confirmed by
`back-hydrolysis in 6 M HCl containing 0.1% stannous chloride
`(Mendex & Lai, 1975).
`The extinction coefficient of CI-INH was determined in
`the following way. CI-INH, at about 4 mg/mL, was dialyzed
`against distilled water adjusted to pH 7.0 with ammonium
`hydroxide. The protein was fully soluble under these condi(cid:173)
`tions, and the 280 nm:260 nm extinction ratio was unaltered
`from that seen in phosphate-buffered saline. The extinction
`of the protein solution at selected wavelengths was measured
`and an aliquot of a standard solution of norleucine added.
`Duplicate aliquots were taken, lyophilized, and hydrolyzed in
`vacuo for 24 h in 6.0 M HCl as described previously. The
`protein concentration of the solution was then determined from
`the hydrolysis data.
`Isolation of Cl-INH. The following procedure has been
`used routinely for plasma volumes of 500-1500 mL.
`Step 1: Removal of Aggregated Material, Fibrinogen, and
`Plasminogen. Freshly drawn human plasma was made 0.01
`M in EDT A and benzamidine by the addition of 0.1 volume
`of a stock solution of 0.1 M EDT A/0.1 M benzamidine, pH
`7.0, and chilled to 4 °C. If fresh frozen plasma was to be used,
`EDT A and benzamidine were added prior to freezing if pos(cid:173)
`sible, otherwise immediately on thawing. All subsequent op(cid:173)
`erations were performed at 4 °C. Solid PEG 4000 was then
`added to the stirred plasma to a final concentration of 5%
`(w jv) and the plasma stirred for a further 1-2 h. The pre(cid:173)
`cipitate, containing aggregated material, factor XIII, and much
`of the fibrinogen, was removed by centrifugation (lOOOOg, 30
`min) and discarded.
`Step 2: Lysine-Sepharose Chromatography. The PEG
`4000 supernatant was applied to a lysine-Sepharose column
`(200-mL bed volume/1000 mL of plasma) equilibrated with
`0.1 ~yhosphate/0.5 M KCl/0.01 M EDTA/0.005 M ben(cid:173)
`zamidme, pH 7 .0, to remove plasminogen. The column was
`
`Page 6 of 11
`
`

`

`c t·INH:
`
`ISOLATION AND STRUCTURE
`
`V 0 L . 2 2 , N 0. 2 I ,
`
`I 9 8 3
`
`5003
`
`~ 2 . 0 0. 2~ n
`JV
`
`40
`
`Fraction
`
`I
`\
`ao
`
`()
`101
`z
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`,.
`5'
`s·
`"
`3
`5 Q,
`0
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`
`120
`
`E
`c
`0
`
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`0
`~
`~
`w
`ID
`u
`u
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`ro
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`~LO 0~1 ~
`<f)
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`2.-P 1.0
`
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`~1.0 OJ5 g
`fi
`ro
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`0
`0
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`25 g
`c.
`c
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`20 ~
`3
`3
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`16 0
`~
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`10 g
`
`50
`
`100
`
`150
`
`Fraction
`fiGURE 1: Elution profile of the post lysine-Sepharose pool on
`DEAE-Sephadex A50. Elution conditions as described in the text.
`(f) A285nm; (•) A6oonm; (•) conductivity; (D) C3; (0) CI-INH; (.0.)
`C4.
`washed with equilibration buffer until the absorbance at 285
`nm of the effluent approached zero; all of the unadsorbed
`protein was pooled.
`Step 3: Chromatography on DEAE-Sephadex A50. A
`DEAE-Sephadex A50 column (400-mL bed volume/1000 mL
`of starting plasma) was equilibrated with 0.02 M phos(cid:173)
`phate/0.1 M NaCl/0.005 M EDTA/0.005 M benzamidine,
`pH 7.0. After the pH and conductivity of the lysine-Sepharose
`effluent were adjusted to those of the equilibration buffer (by
`dilution and addition of 1.0 M HCI), the pool was applied to
`DEAE-Sephadex, and after the column was loaded, it was
`washed with equilibration buffer until the absorbance at 285
`nm was below 0.05. The column was then developed with a
`linear concentration gradient (five column volumes on each
`side) from 0.02 M phosphate/0.005 M EDTA/0.005 M
`benzamidine/0.1 M NaCl, pH 7.0, to 0.02 M phosphate/0.005
`M EDTA/0.005 M benzarnidine/0.4 M NaCl, pH 7.0 (Figure
`1).
`SteP_ 4: Chromatography on Sephadex GJ50 Superfine.
`The C1-INH-containing fractions from the previous column
`were pooled, concentrated to between 1 and 2% of the original
`plasma volume, and dialyzed against 0.02 M phosphate/0.1
`M KCl/0.002 M benzarnidine/0.002 M citrate, pH 7.0. After
`dialysis, the protein was clarified by centrifugation (20000g,
`10 min, 4 °C) and applied to a Sephadex G150 superfine
`column (2000-mL bed volume/1000 mL of starting plasma)
`equilibrated with the above buffer. A load volume of 1-2%
`?f the column bed volume was used. A typical elution profile
`IS shown in Figure 2.
`Step 5: Chromatography on Hydroxylapatite. Cl -INH(cid:173)
`containing fractions from the G 150 effluent were located as
`described previously, pooled, concentrated to between 20 and
`30 mL, and dialyzed against 0.02 M phosphate/0.15 M KCl,
`pH 7 .0. The dialyzed protein was then loaded onto a hy(cid:173)
`droxylapatite column (250-mL bed volume/1000 mL of
`plasma) equilibrated with the same buffer and elution con(cid:173)
`tinued with at least two column volumes of the equilibration
`buffer. This was followed by a gradient from 0.02 M phos(cid:173)
`phate/0.15 M KCl, pH 7.0, to 0.02 M phosphate/1.0 M KCl,
`PH 7.0. Finally, a gradient from 0.02 M phosphate/1.0 M
`I<.Cl, pH 7.Q, to 0.3 M phosphate/1.0 M KCl, pH 7.0, was
`applied. C1-INH-containing fractions (see Figure 3) were
`Pooled, concentrated, and stored frozen at -80 °C.
`
`Results and Discussion
`Isolation of Cl-INH. Because we experienced difficulties
`Particularly wi.th older plasmas and fibrin precipitation, whe~
`
`FIGURE 2: Elution profile of the post DEAE-Sephadex A50 pool on
`Sephadex G150. Elution £Onditions as described in the text. (e)
`A2sonm; (•) A6oonm; (0) Cl-INH.
`
`1.5
`
`Qj
`TI
`~
`0
`
`50
`
`FIGURE 3: . Elution. profile _or the post Sephadex G 150 pool on hy(cid:173)
`droxylapatite: ?lutwn con~Itlons as described in the text. (e) A2sanm;
`(•) con~uctlVlty; (OJ Cl-IN~-1. Inset shows NaDodSOcpoly(cid:173)
`acrylamt~e gel analysis of fractiOns eluting before application of the
`first gradtent. Samples were prepared as described under Materials
`and Methods, and a 10% polyacrylamide gel was used.
`
`using published isolation procedures (Haupt et al., 1970;
`Harpel & Copper, 1975; Reboul et al., 1977), the procedure
`described here was developed. The first two steps (5% PEG
`4000 precipitation and lysine-Sepharose chromatography),
`while achieving little in terms of protein fractionation (see
`Table I), were essential if maximum resolution and recovery
`were to be made in later chromatographic steps. The ad(cid:173)
`vantage of removing plasminogen at an early stage is obvious.
`It is less clear why PEG precipitation is required as consid(cid:173)
`erable amounts of fibrinogen remain in the supernatant. It
`is possible that successful isolation of CI-INH requires the
`removal of factor XIII at this point. A relatively high salt
`concentration (0.5 M KCI) was used during lysine-Sepharose
`chromatography as at lower salt concentrations considerable
`non-ligand-specific adsorption was seen.
`Resolution of CI-INH from the bulk of the plasma proteins
`was achieved by using three conventional chromatographic
`steps. Loading conditions onto DEAE-Sephadex were selected
`such that the albumin was not bound and could therefore be
`washed out of the column prior to appplication of the gradient.
`This was important as it was difficult to remove albumin at
`later stages in the procedure. An additional advantage re(cid:173)
`sulting from these conditi