protein structure and molecular
`enzymology
`
`wl
`8t:.5D3 .
`\1, 705
`no. l .
`
`Page 1 of 10
`
`CSL EXHIBIT 1053
`CSL v. Shire
`
`

`

`Jan .
`
`679 / 1 - B58/1
`
`Feb.
`
`679 /2 - B58/2
`
`March
`
`679/3 - 858 /3
`
`Apr il
`
`680/1 - B59/ 1
`
`May
`
`680/2 - B59 /2
`
`June
`
`680/3 • B59 /3
`
`Jul y
`
`681 / 1 • B60/ 1
`
`Aug .
`
`681/2 • B60/2
`
`Sept.
`
`681/3 = 860/3
`
`Oct.
`
`682 / 1 - 861 / 1
`
`No~ .
`
`682 /2 . 861 /2
`
`Dec.
`
`682/3 • 861 /3
`
`684 / 1 • M95/ 1
`684 /2 • M95/2
`685 / 1 = M96/ 1
`685 /2 • M96/2
`685/3 = M96/ 3
`686/ 1 = M97 / 1
`686/2 • M97/2
`687 / 1 = M98/ 1
`687 /2 • M98/2
`688 / 1 • M99/ 1
`688 /2 • M99 /2
`688/3 = M99/3
`689/1 = M100/ 1
`689/2 • M 100/2
`689 /3 • M100/3
`690/1 = M101/1
`690/2 • M101/2
`691 / 1 • M102/1
`691 /2 • M102 /2
`692/1 • M103/ 1
`692/2 • M 103/2
`692/ 3 • M103/3
`693 / 1 ~ M104/1
`693 /2 = M104/2
`
`696/ 1 • N1 / 1
`
`696/2 = N1 /2
`
`696/3 • N1/3
`
`697 / 1 = N2/ 1
`
`697/2 • N2/2
`
`697/3 = N2 /3
`
`698 / 1 = N3/ 1
`
`698 /2 = N3/2
`
`698 /3 = N3/3
`
`699 / 1 = N4/ 1
`
`699/2 = N4/2
`
`699 /3 • N4/3
`
`700 / 1 = P1 / 1
`700/2 • P1 /2
`701 /1 = P2 / 1
`701/2 • P2 /2
`701 /3 = P2/3
`702 / 1 • P3/ 1
`702 /2 • P3/2
`703 / 1 • P4 / 1
`703 /2 • P4 /2
`704 / 1 = PS / 1
`704 /2 • PS /2
`704 /3 = PS /3
`705 / 1 • P6/ 1
`705 /2 • P6/2
`705 /3 • P6/3
`706 /1 • P7/1
`706/2 = P7 /2
`707 / 1 • P8 / 1
`707 /2 = PS /2
`708 / 1 - P9 / 1
`708 / 2 . P9 /2
`708/3 • P9 /3
`709 / 1 = P10/ 1
`709 /2 • P10/2
`
`711/3 • L59/3
`
`716/2 • G98/2
`
`71 2/2 = L60 /2
`
`713/1- L61 / 1
`
`713 /2 = L61/2
`
`716 /3 • G98/3
`712 / 1 = L60/ 1 717 / 1 • G99/ 1
`717/2 • G99/2
`717 /3 = G99/3
`712/3 = L60/3 718 / 1 • G100/ 1
`718 /2 = G100/2
`719 / 1 • G101 / 1 721 /3 • C2/3
`
`PUBLICATION SCHEDULE FOR 1982
`
`BBA is published according to a volume-numbering scheme that embraces all sections of the journal. In addition,
`each regular section has its own sequential volume-num.bering system, running parallel to the overall BBA scheme
`set out below.
`
`BIO-
`BIO·
`ENERGETICS MEMBRANES
`
`LIPIDS
`PROTEIN
`GENE
`STRUCTURE STRUCTURE AND LIPID
`AND
`METABOLISM
`AND
`EXPRESSION MOLECULAR
`ENZYMOLOGY
`
`GENERAL
`SUBJECTS
`
`MOLECULAR
`CELL
`RESEAR CH
`
`710/ 1 • LSB / 1 714/1 • G96/ 1
`
`710/2 •· L58 /2
`
`714/2 • G96/2
`
`720/ 1 • C1 / 1
`
`71 0 /3 = L58 /3
`
`714 /3 • G96/3
`715 / 1 • G97/ 1 · 720 /2 = C1/2
`711/1 • L59 / 1 715 /2 = G97/2
`716/ 1 • G98/ 1
`
`711 /2 = L59/2
`
`720 /3 = C1/3
`
`720/4 = C1/4
`
`721 /1 • C2 / 1
`
`721 /2 • C2 /2
`
`719 /2 • G101 /2
`
`713 / 3 • L61/3 719 /3 = G101 /3
`
`721 /4 • C2/4
`
`BBA REVIEWS ON BIOENERGETICS. In addition to the 4 regular volumes of the BIOEN ERGETI CS section. 1 volume of I
`BBA REVIEWS ON BIOENERGETICS (Vo l. 683 • BR10l w;ll be pub!;shed .
`BBA REVIEWS ON BIOMEMBRANES . In addition to the 10 regular volumes of th e BIOMEMBRAN ES section, 1 volume of
`BBA REVIEWS ON BIOMEMBRANES (Vol . 694 • MR11} w;ll be pubnshed .
`BBA REVIEWS ON CANCER. Included in the total number of volumes, 1 volume of BBA REVIEWS ON CANCER !Vol. 695 =
`CA9) will be published .
`
`<0 Elsevier Biomedical Press, 1982
`Printed in The' Netherlands
`
`All rights reserved . No part of this publication may be reproduced, stored in a retrieval system , or transmitted, in any form or by any
`means, electronic, mechanical, photocopying, recording or otherwise without the prior permission of the copyright owner.
`Submission to this journal of a paper entails the author's irrevocable and exclusive authorization of the publisher to collect any sums
`or considerations for copying or reproduction payable by third parties (as mentioned in article 17 paragraph 2 of the Dutch
`Copyright Act of 1912 and in the Royal Decree of June 20, 1974 (S. 351) pursuant to article 16b of the Dutch Copyright Act of 191 2)
`andj or to act in or out of Court in connection therewith .
`
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`
`Page 2 of 10
`
`

`

`Biochim ica et Biophysics Acta 705(2)
`protein Structure and Molecular Enzymology P6(2)
`
`July 26, 1982
`
`CONTENTS
`
`(Cited in: Current Contents Life Sciences - Biological Abstracts - Chemical Abstracts -
`Excerp ts Medica)
`
`Index Chemicus -
`
`Index Medicus -
`
`purification and characterization of extracellular poly(cid:173)
`amine o xidase produced by Penicillium sp. No. P0-1
`(BBA 31226)
`by Y. K obayashi and K. Horikoshi (Saitama, Japan) . . .
`
`133
`
`transfer in peptides. Intramolecular radical
`Charge
`transformations involving methionine, tryptophan and
`tyrosine (BBA 31231)
`by W.A. Protz, F. Siebert, J . Butler, EJ. Land, A. Menez
`and T. Montenay-Garestier (Freiburg, F.R.G., Manches-
`ter, U.K. and Gif-sur- Yvette and Paris, France) . ...... .
`
`Charge transfer between tryptophan and tyrosine in
`proteins (BBA 31233)
`by J . Butler, E.J. Land, W.A. Protz and A.J. Swallow
`(Manchester, U.K. and Freiburg, F.R.G.) . ... . .
`
`C haracterization of the proteolytic activity firmly at(cid:173)
`tached to yeast phosphoenolpyruvate c arboxykinase
`(BBA 31227)
`by I. Beck, M. MUller and H. Holzer (Munich and Frei-
`burg. F.R . G.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Isolation and characterization of glycopeptides of hu(cid:173)
`man transcortin (BBA 31224)
`I. V. Mat(cid:173)
`by O.A. Strei'Chyonok, G. V. Avvakumov,
`veentseva, L. V. Akhrem and A.A. Akhrem (Minsk,
`U.S.S. R.).. . ..... . ...... ... ... . .... ... ... . . . . . .
`
`Isolation and purification of rat acute-phase a 2 -macro(cid:173)
`g lobulin (BBA 31229)
`by D.E. Panrucker and F.L. Lorscheider (Calga;y,
`Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Radioimmunoassay of rat acute-phase a 2 -macroglobu(cid:173)
`lin (BBA 31230)
`by D.E. Panrucker and F.L. Lorscheider (Calgary,
`Canada) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Purification and partial characterization of two forms of
`urinary trypsin inhibitor (BBA 31 235)
`by Y. Tanaka. S. Maehara, H. Sumi, N . Toki, S. Moriyama
`and K . Sasaki (Tokyo, Shimane, M iyazaki and
`Hiroshima, Japan) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`The effect of aging on rat liver phosphoglycerate kinase
`and c omparison with the muscle enzyme (BBA 31237)
`by L.S. Hiremath and M . Rothstein (Buffalo, NY, U.S.A.)
`
`Purification and properties of the pyruvate dehydro(cid:173)
`genase complex from Salmonella typhlmurium and for(cid:173)
`mation of hybrids with the enzyme complex from
`Escherichia coli (BBA 31239)
`by R . Seckler, R. Binder and H. Bisswanger (Tabingen,
`F .R.G.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`labeling of membrane-bound porcine
`Photoaffinity
`aminopeptidase N (BBA 31250)
`by D. Gratecos, L. Varesi, M . Knibiehler and M . Semeriva
`13~___-c'Marseilles, France) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`Demonstration that bovine erythrocyte cytochrome bs
`is the hydrophilic segment of liver m icrosomal cyto(cid:173)
`chrome b5 (BBA 31240)
`1&o-- by S.R. Slaughter, C.H. William s, Jr. and D.E. Hultquist
`(Ann Arbor, Ml, U.S.A.)
`. . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Effect of vanadate on the formation and stability of the
`phosphoenzyme forms of 2,3-bisphosphoglycerate-de(cid:173)
`pendent phosphoglycerate mutase and ot phos(cid:173)
`phoglucomutase (BBA 31246)
`by J . Carreras, F. Climent. R . Bartrons and G. Pons
`(Barcelona, Spain) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`The isoelectric focusing of human thyroglobulin (BBA
`31236)
`by C. Davoli, S. Grimaldi, G. Rusca, M . Andreoli and H.
`Edelhoch (Rome, Italy) . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Isolatio n and partial characterization of prochymosin
`and chymosin from cat (BBA 31244)
`by T. Jens en, N.H . Axelsen and B. Foltmann
`(Copenhagen, Denmark) . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Recombination kinetics following nanosecond laser
`photolysis of carbonmonoxyhaemoglobin (BBA 31238)
`by R . Catterall, D.A. Duddell, R.J. Morris and J. T.
`Richards (Salford, U.K.) . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`C ircular dichroism studies on a 2 -antiplasmin and its
`interactions with plasmin and plasminogen (BBA 31 264)
`by T. Nilsson, I. SjOholm and B. Wiman (Umell and
`Uppsala, Sweden) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
`
`Purification and characterization of human C1-esterase
`Inhibitor (BBA 31265)
`by T. Nilsson and B. Wiman ( Umell, Sweden) . . . . . . . . .
`
`163
`
`167
`
`17 4
`
`184
`
`192
`
`200
`
`210
`
`218
`
`228
`
`238
`
`243
`
`249
`
`257
`
`264
`
`211
`
`INFORMATION FOR CONTRIBUTORS TO BIOCHIMICA ET BIOPHYSICA ACTA
`
`The 1982 edition of thla booklet, offering detailed guidance to authors, Ia available free of charge from the BBA Editorial
`Secretariat, P.O. Box 1345, 1000 BH Amsterdam, The Netherlands. It Ia alao published aa Blochlm. Blophya. Acta 715 (1982) 1-23.
`
`continued
`
`Page 3 of 10
`
`

`

`Tropomyosin structure and intermolecular contacts in
`Mg paracrystals (BBA 31222)
`by S.D. Golladay (Chicago, IL, U.S.A.)
`
`. . . . . . . . . . . . . .
`
`277
`
`Inhibitory action of proline-containing peptides on Xaa(cid:173)
`Pro-dipeptidylaminopeptidase (BBA 30023)
`by M. Harada, K.M. Fukasawa, K. Fukasawa and T.
`Nagatsu (Shiojiri and Yokohama, Japan) . . . . . . . . . . . .
`
`BBA Reports
`
`Titles of related papers in other sections
`
`Arrhenius plot characteristics of membrane-bound 3-
`hydroxy-3-methylglutaryl coenz~me A reductase from
`the latex of Hevea brasiliensis (BBA 30018)
`by A.B. Sipat (Selangor, Malaysia) . . . . . . . . . . . . . . . . .
`
`284
`
`. . •. , ., . . .
`"-.··.
`
`288
`
`291
`
`BIOCHIMICA ET BIOPHVSICA ACTA
`International Journal of Biochemistry and Biophysics
`Founded In 1947 by H.G.K. Weatenbrlnk, Managing Editor, 1947- 1964
`
`e EIMYier Biomedical Press, 1982
`Printed In The Netherlands
`
`All rtghta reserved. No part of this publication may be reproduced, stored In a retrieval ayatem, or transmitted, In any form o r by
`means, electronic , mechanical, photocopying, recording or otherwlae without the prior permission of the copyright
`Submission to this journal of a paper entails the author's irrevocable and exclusive authorization of the publisher to·collect any
`or considerations tor copying or reproduction payable by third parties (as mentioned In article 17 paragraph 2 of the Dutch
`Copyright Act of 1812 and In the Royal Decree of June 20, 1974 (S. 351) pursuant to article 16b of the Dutch Copyright Act of 1912}
`and for to act In or out of Court In connection ther-lth.
`
`Page 4 of 10
`
`

`

`Biochimica et Biophysica Acta, 705 (1982) 271-276
`Elsevier Biomedical Press
`
`BBA 31265
`
`271
`
`PURIFICATION AND CHARACfERIZATION OF HUMAN Cl-ESTERASE INIDBITOR
`
`TORBJORN NILSSON and BJORN WIMAN •
`
`Department of Clinical Chemistry, Umeti University Hospital, S-901 85 Umeti (Sweden)
`
`(Received December 16th, 1981)
`
`Key words: Cl-esterase; Disulfide; Esterase inhibitor; Complement; (Human)
`
`A new purification method for Cl-esterase inhibitor is described, which is essentially a three-step procedure:
`precipitation with poly(ethylene glycol), chromatography on DEAE-cellulose and· hydrophobic interaction
`chromatography on hexyl-Sepharose. The final product is a single-chain glycoprotein with a molecular weight
`of about 100000 and NH 2 -terminal asparagine. The molecule is fully active as judged by complex formation
`with Cis. Two of its three disulphide bridges can be easily reduced and S ~carboxymethylated under ·
`non-denaturing conditions without loss of activity. However, at high dithioerythritol concentration the third
`disulphide bridge is also cleaved and accompanied by loss of the activity, indicating that this disulphide bridge
`is involved in maintaining the conformation around the reactive site in the inhibitor.
`
`Introduction
`
`Human plasma contains several protease inhibi(cid:173)
`tors of well-known functions. Among these, the
`Cl-esterase inhibitor is important in the regulation
`of the complement system [ 1] and possibly also in
`the kininogen system [2] and the intrinsic fibrinol(cid:173)
`ysis [3]. Several purification methods for the Cl(cid:173)
`esterase inhibitor have been described [1,4-8], al(cid:173)
`though few seem to give fully satisfactory prepara(cid:173)
`tions suitable for functional studies. In ·order to
`perform such studies we have therefore developed
`a simple method for purification of cT -esterase
`inhibitor which can be operated on a large scale
`and results in a high yield of pure product, with
`properties as close to the native molecule as possi(cid:173)
`ble.
`
`Materials and Methods
`
`Human plasma. Freshly frozen citrated human
`plasma was obtained from the Blood Centre of
`
`• To whom correspondence should be addressed.
`
`Oi67-4838j 82j0000-0000j $02.75 © 1982 Elsevier Biomedical Press
`
`UmeA University Hospital (courtesy of Dr. B.
`Cedergren) ..
`Cis. This was prepared essentially according to
`the method of Chapuis et al. [9). The final product
`was a homogeneous protein as established by
`SDS-polyacrylarnide gel electrophoresis, all of
`which could form a stable complex with Cl(cid:173)
`esterase inhibitor, if this was added in excess.
`Reagents. Hexyl-Sepharose was obtained by
`coupling 100 m1 1M hexylamine (Kebo AB,
`Stockholm, Sweden), adjusted to pH 9 with hydro(cid:173)
`chloric acid, to 1000 m1 CNBr-activated Sepharose
`4B [10]. Sepharose 4B® and DEAE-Sephacel® were
`from Pharmacia Fine Chemicals (Uppsala,
`Sweden). Polybrene was from Aldrich (Beerse, Bel(cid:173)
`gium) and the chromogenic tri-peptide-p-nitroani(cid:173)
`lide substrate S-2400 was a gift from Kabi peptide
`Research (Molndal, Sweden, courtesy of Dr. Lars
`Mellstam). All reagents and chemicals used were
`of analytical grade.
`N H2-termina/ amino acid determination. This was
`performed by manual Edman degradation [ 11],
`and the phenylthiohydantoins were identified by
`thin-layer chromatography as described previou,sly
`[ 12].
`
`Page 5 of 10
`
`

`

`272
`
`Amino acid analysis. The samples were hydro(cid:173)
`lyzed in constant boiling HCl in vacuo for 24, 48
`and 72 h. Analysis was performed on an LKB
`amino acid analyzer according to the procedures
`given by the manufacturers.
`Immunochemical analysis. Rabbit antiserum to
`the CI-esterase inhibitor was raised by injection of
`the purified protein mixed with Freund's complete
`adjuvant. Booster doses were given together with
`Freund's incomplete adjuvant when the specific
`antibody titers were decreasing.
`Electroimmunoassay was performed according
`to the method of Laurell [ 13] and crossed im(cid:173)
`munoelectrophoresis essentially as described by
`Clarke and Freeman [14).
`Determination of Cis and CI-esterase inhibitor
`concentrations. The Cis concentration was de(cid:173)
`termined from its absorbance at 280 nm, using an
`A~onm of 9.5 [15].
`The Cis activity was measured using the chro(cid:173)
`mogenic substrate S-2400: a 140 nM solution of
`Cis gave a ~A410 of 0.043/ min at a substrate
`.concentration of 0.63 mM in 0.1 M sodium phos(cid:173)
`phate buffer, pH 7.3, at 25°C.
`CI-esterase inhibitor concentrations were de(cid:173)
`termined spectrophotometrically using an A~~Onm
`of 5.0 [1), or by titration against purified Cis in
`the following way: 25 ~tl of a CI-esterase inhibitor
`sample was incubated for 10 min with 25 ~tl Cis
`(final concentration 140 nM) in a cuvette. Then
`0.7 ml 0.1 M phosphate buffer, pH 7.3, and 200 ~tl
`3 mM S-2400 were added and residual Cis activity
`was measured from the ~A41onm• as described
`above. The CI-esterase inhibitor could then be
`calculated from the decrease in ~A41onm·
`Polyacrylamide gel electrophoresis. SDS-poly(cid:173)
`acrylamide gel electrophoresis according to the
`method of Weber and Osborn [16] was performed
`on non-reduced and reduced samples, as previ-
`ously described [ 17).
`Reduction and S-carboxymethylation. Reduction
`of the CI-esterase inhibitor was performed using a
`protein concentration of about 2 mgj ml. Di(cid:173)
`thioerythritol was added to final concentrations of
`0.03- 200 mM. For reductions at non-denaturating
`conditions, a 0.1 M sodium phosphate buffer, pH
`7.3, was used. For reductions at denaturing condi(cid:173)
`tions, the same buffer, containing 6 M guanidinium
`chloride, was used. After incubation for 30 min at ·
`
`~
`
`25°C in nitrogen athmosphere, [3H)ICH 2COONa
`was added to a final concentration of 80 mM, and
`incubation was continued in the dark for another
`5 min. To the 200 mM dithioerythritol sample, 450
`mM ICH 2C00Na was used. The labelled protein
`was isolated by gel filtration on Sephadex G-50
`columns (0.8 cm2 X 9 em) equilibrated with 0.1 M
`sodium phosphate buffer, pH 7.3. Control samples
`that no di(cid:173)
`were similarly
`treated, except
`thioerythritol was added. The separated protein
`samples were analyzed by radioactivity measure(cid:173)
`ment (Packard Tri-carb liquid scintillator) and
`protein determination by A280 or amino acid anal(cid:173)
`ysis after acid hydrolysis. As a reference protein,
`a 2-antiplasmin (10 mgj ml) was treated in the same
`way in order to obtain the specific radioactivity of
`the iodoacetic acid, since it is known that this
`protein contains three disulphide bridges and no
`free SH group [18,19).
`
`Results
`
`Purification of CI-ester.ase inhibitor
`All procedure were carried out at + 5°C. About
`1 litre of plasma depleted in plasminogen by treat(cid:173)
`ment with lysine-Sepharose (20] was thawed, made
`20 mM in EDT A, and polybrene was added to a
`final concentration of 0.6 gj l. The plasma was
`subsequently diluted with an equal volume of PEG
`6000 (12%, wj v). After stirring for 30 min, the
`precipitate was removed by centrifugation, and to
`the clear supernatant were added about 300 m1 of
`DEAE-Sephacel®, equilibrated with 0.04 M phos(cid:173)
`phate buffer, pH 7.0.
`After stirring for 30 min, the gel was washed on
`a Buchner funnel with about 15 litres of the same
`phosphate buffer. The gel was then degassed and
`packed in a column (5 X 15 em), and subsequently
`eluted employing a linear gradient of NaCl from 0
`to 0.3 M (total volume 1800 ml) in the same phos(cid:173)
`phate buffer (Fig. 1 ).
`The CI-esterase inhibitor concentration in the
`eluted fractions was determined by electroim(cid:173)
`munoassay. Generally it was eluted in the 0.15-
`0.18 M NaCl range. The fractions containing anti(cid:173)
`gen were pooled, concentrated to about 30 ml and
`made 0.4 M in (NH 4 hS04 by addition of an ap(cid:173)
`propriate amount of the solid substance.
`The concentrated DEAE-Sephacel® pool was
`
`Page 6 of 10
`
`

`

`4.0
`
`E 3.0
`c
`~
`c
`QJ 2.0
`u
`c
`_g
`5
`"'
`.D
`<{
`
`1.0
`
`15]
`E
`-~
`.....
`d
`1.0 ~
`QJ
`u
`5
`o.5 a
`
`u
`
`500
`Elution volume lmll
`
`1000
`
`Fig. I. Elution of the DEAE-Sephacel column by a linear
`gradient from 0.04 M sodium phosphate buffer, pH 7.0, to the
`same buffer containing 0.3 NaCI. A280 ( - - ) ; CT-esterase
`inhibitor (CIN) concentration according to Laurell electro(cid:173)
`phoresis (----- -).
`
`then applied to a column of hexyl-Sepharose (5 X
`20 em), equilibrated with 0.4 M (NH 4 hS04 in
`0.04 M phosphate buffer, pH 7.0. The Cl-esterase
`inhibitor passed through the column unadsorbed,
`whereas the contaminating proteins were either
`adsorbed or delayed (Fig. 2).
`The first peak containing the C)-esterase in(cid:173)
`hibitor was pooled as indicated, and subsequently
`dialyzed against 0.1 M sodium phosphate buffer,
`pH 7.3, in the cold. The solution was thereafter
`concentrated to give a final concentration of about
`3 mg proteinjml and stored frozen at - 80°C.
`Typically about 80-100 mg of the purified CT-
`
`0.4
`
`N
`
`0.3
`
`273
`
`esterase inhibitor were obtained from 1 litre
`plasma.
`
`Characterization of the purified CI-esterase inhibi(cid:173)
`tor
`
`Homogeneity. The product obtained after the
`hexyl-Sepharose step is typically a single-chain
`protein which is more than 95% pure as shown by
`SDS-polyacrylamide gel electrophoresis on re(cid:173)
`duced and non-reduced samples (Fig. 3). On com(cid:173)
`parison with reference proteins its molecular weight
`was estimated to about 100000. On crossed im(cid:173)
`munoelectrophoresis, using a rabbit antiserum
`raised against the purified protein, only one pre(cid:173)
`cipitation arc is observed for the purified C)(cid:173)
`esterase inhibitor, as well as for human pooled
`plasma (Fig. 4). Within experimental error the
`electrophoretic mobility of the pure product is
`identical or very similar. to the native CT-esterase
`inhibitor in plasma. No signs of this inhibitor in
`complex with proteases such as Cis or CTr have
`been detected in normal plasma samples.
`The amino acid composition (Table I) is in ex(cid:173)
`cellent agreement with that reported by Haupt et
`al. [4] for most amino acids. The only exception is
`tyrosine, where our figure is considerably lower.
`The amino acid composition reported by Harpel
`[1] is, however, quite different. On NH 2-tei:minal
`amino acid analysis, about 1 mol asparagine per
`mol protein was found. No other amino acid could
`be detected.
`Activity. Titration of the purified Cl -esterase
`inhibitor with Cis resulted in an inverse linear
`relationship between the amount of Cl-esterase
`inhibitor added and remaining Cis activity. A 10%
`molar excess of the inhibitor was sufficient to
`abolish the cT s activity.
`SDS-polyacrylarnide gel electrophoretic analy(cid:173)
`sis of the reaction mixture obtained using Cis in
`
`E c
`2
`-a
`QJ
`u 0.2
`c
`Cl
`.D
`'-iil 0.1
`
`.D
`<{
`
`700
`600
`500
`400
`Elution volume lmll
`
`800.
`
`Fig. 2. Hydrophobic interaction chromatography on hexyl-Sep(cid:173)
`harose. The pooled fraction from the DEAE-Sephacel column
`was applied to the hexyl-Sepharose column equilibrated with
`0.04 M sodium phosphate buffer containing 0.4 M (NH 4 hS04 •
`The first peak consists of essentially pure Cl-esterase inhibitor.
`
`0
`
`b
`
`Fig. 3. SDS-polyacrylarnide gel electrophoretograms of the
`purified CI-esterase inhibitor: (a), reduced sample; (b), non-re(cid:173)
`duced sample.
`
`Page 7 of 10
`
`

`

`274
`
`Fig. 4. Crossed immunoelectrophoresis of (a) purified C)(cid:173)
`esterase inhibitor, (b) normal human plasma. The second di(cid:173)
`mension was run into a gel containing I% (v j v) rabbit anti(cid:173)
`C)-esterase inhibitor.
`
`exces~ revealed that all of the CT-esterase inhibitor
`reacted to form a complex with the CT s light
`chain. When the CT-esterase inhibitor was in ex(cid:173)
`cess all of the CTs was converted to the enzyme-in(cid:173)
`hibitor complex, indicating that both the inhibitor
`and the CTs preparations fully retain their activi(cid:173)
`ties.
`Stability. A fresh preparation of CT-esterase
`inhibitor, dialyzed against 0.1 M phosphate buffer,
`pH 7.3, was divided intq aliquots and kept at
`+5°C, -20°C or -80°C. Sodium azide (0.04%)
`was added as preservant in the + 5 °C aliquots. At
`different times, ranging between 0-50 days, sam(cid:173)
`ples were checked for inhibitory activity against
`Cis. At -80°C or at +5°C no decline in activity
`could be detected. However, at - 20°C a small but
`steady decline in activity was observed, amounting
`
`TABLE I
`AMINO ACID COMPOSITION OF THE PURIFIED PRE(cid:173)
`PARATION OF C)-ESTERASE INHIBITOR
`
`The figures represent the mean value of three determinations.
`
`Amino acid
`
`Aspartic acid
`Threonine •
`Serine •
`Glutamic acid
`Proline
`Glycine
`Alanine
`Cysteine •
`Valine
`
`11mol
`IOOmg
`
`Amino acid
`
`11mol
`100 mg
`
`56.5
`59.9
`52.5
`61.0
`42.6
`17.0
`38.0
`5.5
`37.1
`
`Methionine
`Isoleucine
`Leucine
`Tyrosine
`Phenylalanine
`Histidine
`Lysine
`Arginine
`
`14.5
`19.8
`62.7
`8.3
`29.2
`9.0
`36.9
`16.0
`
`• Extrapolated values.
`
`to about 50% during the observation period.
`The disulphide bridges in CI-esterase inhibitor.
`Amino acid analysis has indicated six cysteines in
`the molecule (Table 1), thus forming three dis(cid:173)
`ulphide bridges. This has been investigated further
`by reduction of the CT-esterase inhibitor with di(cid:173)
`thioerythritol in the presence of 6 M guanidinium
`chloride, followed by S-carboxymethylation with
`3H]CH 2COONa. Using a 2-antiplasrnin as a
`[
`~tandard, known
`to possess
`three disulphide
`bridges [19], we found 5.9 cysteines in the cT(cid:173)
`esterase inhibitor. The small radioactivity incorpo(cid:173)
`rated nonspecifically prior to reduction, corre(cid:173)
`sponding to about 0.8 residues, is then already
`subtracted. Conclusively, the CT-esterase inhibitor
`contains three disulphide bridges.
`Partial reduction of the CT-esterase inhibitor
`under non-denaturiog conditions using different
`dithioerythritol concentrations
`followed by
`S-carboxymethylation with [3H]ICH 2C00Na is
`shown in Fig. 5. Two of the three disulphide bridges
`seem to be easily cleaved even at 3 mM di(cid:173)
`thioerythritol under non-denaturing conditions.
`However, to cleave the third disulphide bridge the
`dithioerythritol concentration has to be increased
`to 200 mM. Included in Fig. 5 are also the results
`from activity measurements of the reduced CT(cid:173)
`esterase
`inhibitor samples. Reduction and
`S-carboxymethylation of the two easily reduced
`disulphide bridges are performed without loss of
`
`Page 8 of 10
`
`

`

`activity. However, cleavage of the third disulphide
`bridge results in almost complete loss of activity
`(Fig. 5).
`
`Discussion
`
`Many of the previously published purification
`procedures for CT-esterase inhibitor are tedious
`and result in low yields and varying amounts of
`degraded forms of the inhibitor. The method re(cid:173)
`cently published by Reboul et al. [5] is simple, but
`in our hands it only gave a 50%-pure material with
`several contaminants at low concentrations. There(cid:173)
`fore the present method seems to be superior in its
`simplicity, high yield and quality of the final prod(cid:173)
`uct. A yield of 80- 100 mgj litre plasma represents
`about 45-55%, assuming a concentration of 180
`mg/ 1 in normal plasma [1]. The present method
`can, furthermore, easily be operated on a large
`scale.
`The purified protein is a single-chain glycopro(cid:173)
`tein with a molecular weight of about 100000, and
`asparagine is found to be its NH 2-terminal amino
`acid. The purified preparation has electrophoretic
`mobility in the a 2 region identical to that of the
`CT-esterase inhibitor in plasma, as judged by
`crossed immunoelectrophoresis. It is also fully ac(cid:173)
`tive, as judged by titration against CTs or analysis
`of a mixture of CT-esterase inhibitor and CTs in
`molar excess by SDS-polyacrylarnide gel electro-
`
`z
`4
`Ci
`~
`:X:
`~
`~ 2
`
`100
`
`so
`
`~
`~
`·;;
`:0:::
`::!
`z
`Ci
`
`DTE Concentration ( mMl
`
`100
`
`1000
`
`Fig. 5. Reduction of Cl-esterase inhibitor (CIN) by different
`concentrations of dithioerythritol (DTE)
`followed by
`S-carboxymethylation with [3 H]ICH 2C00Na. The number of
`S-carboxymethylcysteines were calculated from the specific
`radioactivity (e--e). Included in the figure are also the
`results from activity measurements ( 0 ).
`
`275
`
`phoresis. If kept frozen at - 80°C the material is
`stable for months, but not if frozen at - 20°C.
`Lyophilization always results in loss of appreciable
`amounts of activity.
`Recently we reported [19,21] that two of the
`three disulphide bridges in a 2-antiplasmin could
`be reduced and S-carboxymethylated under non(cid:173)
`denaturating conditions without affecting
`the
`inhibitory activity. Cleavage of the third dis(cid:173)
`ulphide bridge could only be performed in the
`presence of denaturing agents and was accompa(cid:173)
`nied by complete loss of activity [19,21]. In the
`present study, similar results are obtained with
`CT-esterase inhibitor, although
`the
`third dis(cid:173)
`ulphide bridge in this case can be cleaved if a high
`concentration of dithioerythritol is used. This still
`leads to inactivation of the molecule, indicating
`that the 'stable' disulphide bridge is indeed im(cid:173)
`portant in maintaining the crucial conformation
`around the reactive site of the inhibitor. The simi(cid:173)
`lar behaviour of the disulphide bridges in a 2-anti(cid:173)
`plasmin and CT-esterase inhibitor may indicate a
`structural relationship between these molecules.
`This will be further elucidated by amino acid
`sequence analyses, which are already in progress.
`
`Acknowledgements
`
`Financial support has been obtained from the
`Swedish Medical Research Council (project No.
`05193) and
`the Lions Research Foundation,
`Department of Oncology, Umea University (pro(cid:173)
`jects 179 ; so and 206/ 81 ). The expert technical
`assistance of Ms. Asa Almquist and Mr. Tord
`Arvidsson is gratefully acknowledged.
`
`References
`
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`2 Gigli, 1., Mason, J.W., Colman, R.W. and Austen, K.F.
`(1970) J. Immunol. 104, 574- 581
`3 Kluft, C. ( 1977) Haemostasis 6, 351 - 369
`4 Haupt, H., Heimburger, N ., Kranz, T. and Schwick, H.G.
`(1970) Eur. J. Biochem. 17, 254-261
`5 Reboul, A., Arlaud, G.J., Sim, R.B. and Colomb, M.G.
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`6 Harpel, P.C. and Cooper, N :P. (1975) J. Clin. Invest. 55,
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`7 Nagati, K., l ida, K. and lnai, S. (1974) Int. Arch. Allergy
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`8 Anderson, W.H.K., Smith, J.K. and Fothergill, J.E. (1975)
`Biochem. Soc. Trans. 3, 933-934
`
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`munochemistry 14, 313-317.
`10 Hjertim, S. (1981) Methods Biochem. Anal. 27, 89-108
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`(Needleman, S.B., ed.), pp. 232-279,
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`
`Page 10 of 10
`
`