w I
`51503
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`pmtcin structure and molecular
`enzymology
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`K RECE’
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`‘ AUG 6 3982
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`w-‘m mm! ucso !
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`Page 1 of 10
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`CSL EXHIBIT 1053
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`

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`PUBLlCATION SCHEDULE FOR 1982
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`BBA is published according to a volume-numbering scheme that embraces all sections at the journal. In addition,
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`each regular section has its own sequential volume-numbering system, running parallel to the overall EBA scheme
`set out below.
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`610‘
`BID»
`GENE
`PROTEIN
`LIPIDS
`GENERAL
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`ENERGETICS MEMBRANES
`STRUCTURE
`STRUCTURE AND LIPID
`SUBJECTS
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`AND
`METABOLISM
`AND
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`EXPRESSION MoLECULAR
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`_,__
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`J
`ENZYMOLOGY
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`684/1 = mes/1
`700/1 = 171/1
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`696/1 N1/1
`679/16 855/1 68“? g Mes/2
`700/2 = 171/2
`714/1 Gas/1
`710/1 — 1.58/1
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`679/2 = 358/2 685“ = M96“
`696/2 = 111/2
`70‘“ = P2"
`‘71012 =‘ Lea/2 7W2 ‘ 696/2
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`701/2 = 72/2
`685/2 = M86/2
`714/3 : 696/3
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`685/3 = Mes/3
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`701/3 = 192/3
`7 —
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`679/3
`066/3 606/1= 1107/1
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`696/3— N1/3 “—1702“:113/1
`710/3 — mm 715” = (387/1 _ 720/2 =01/2
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`686/2 = M97/2
`702/2 . P3/2 ‘—*
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`059/1
`680/1
`687I1= M98“
`697/1 mm 703“: P4/1
`711/1
`L59/1
`716/2
`007/2 ‘
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`680/2 = 1359/2 687/2 = M86/2
`711/2 = L59”
`697/2 = 112/:
`703/2 = P4/2
`716/1 - 698/1
`1' 720/3 - c1/3
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`688/1= M99/1
`704/1= 75/1
`716/2 = 69m ‘1
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`704/2 = P5/2
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`688/2 = M99/2
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`697”
`112/3
`680/3 ' ”59/3 688/3 = 1199/:
`704/3 = r5/3
`“”3 ' L59“! 716/3 : 698/3
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`688/1 = M100/1
`705/1 = P6/1
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`689,2 : woo/2 658/1_ 113/1
`705/? = P6]?
`712/1 - 1.60/1
`681/1 7 1160/1
`717/1 - 099/1
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`7?1/1= (32/1
`705/3 = 95/3
`689/3 = M1005
`A
`71712 = ($99!?
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`698/2
`“3’2
`706/1 = 177/1
`68‘” 350/2 690/1 = M101/1
`717/3 = (599/3
`“2’2 ' Lea/2
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`706/2 = r712
`690/2 = M101/2
`7
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`707/1= Pan
`681/3 - 360/3 691/1 = M102”
`698/3
`1113/3
`712/3 - 1.60/3
`710/1— (1100/1
`721/2 - 62/2
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`707/2 = P8]?
`“—K
`z
`691/2 = M102/2
`718/2 - 6100/2
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`1114/1
`682/1
`361/1 692” = M103/1
`699/1
`7013/1 x 119/1
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`713/1 - 1.61/1 [—719/1_ 6101/1
`721/3 _ c2/3
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`692/2 = M103/2
`708/2 = Pea/2
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`1
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`699/2
`1114/2
`70m = P9/3
`682/2
`66 [2
`692/3 : M103/3
`713/2 L61/2
`719/2 . 6101/2
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`693/1 - M10411
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`709/1 = Pic/1
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`861/3 693/2 . M104/2 698/3 — 114/3
`i011:,
`682/3
`709/2 ; P1012
`719/3 7 6101/3
`713/3 - 1.61/3
`721/4 . 02/3
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`BEA REVIEWS ON BIOENERGETICS,
`In additlon [0 [he 4 regular volumes 011712 BIOENERGETICS section,
`'I volume all
`BEA REVIEWS on BlOENERGETlCS (Vol. 60:1 = BR‘lOI Will be published.
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`EBA REVIEWS ON BIOMEMEHANES. In addition to the 10 regular volumes 91 the BIOMEMBRANES section, 1 volume 0'
`BSA REVIEWS ON BIOMEMBRANES (VDI. 694 = MR1” Will be published.
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`BEA REVIEWS ON CANCER. included in the total number of volumes, 1 volume of BEA REVIEWS 0N CANCER (Vol, 695 :
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`CR9) will be published.
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`[—
`_—u
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`MOLECULAR
`CELL
`RESEARCH
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`720/1 = c1/1
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`720/4 = C114
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`Jan,
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`Feb.
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`March
`-
`April
`my
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`4"”
`July
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`“9'
`Sept.
`0:1
`gNoy
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`© Elsevier Biomedical Press, 1982
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`Printed In The Netherlands
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`All rights reserved. No part of this publication may be reproduced. stored in a retrieval system, or transmitted. in any form or by any
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`means. electronic. mechanical, photocopying,
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`Submission to thlsjournal of a paper entails the author's irrevocable and exclusive authorization of the publisher to collect any sums
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`or considerations tor copying or reproduction payable by third parties (as mentioned in article 17 paragraph 2 of the Dutch
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`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 1912)
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`condition that the copier pays through the Center the per—copy tee stated in the code on the first page of each article for copying
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`not extend to other kinds of copying. such as for general distribution. resale. advertising and promotion purposes, or for creating
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`new collective works. Special written permission must be obtained from the publisher tor such copying.
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`copyright of the article to the publisher. This transfer will ensure the widest possible dissemination of information under the US.
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`Copyright Law.
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`Page2 of10
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`Page 2 of 10
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`

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`Bmcnimica el Blophysica Acta 705(2)
`pmrern Structure and Molecular Enzymology P6(2)
`
`CONTENTS
`
`July 26, 1992
`
`(cued in: Current Contents Lila Sciences — Biological Abstracts — Chemical Abstracts — Index Chemicua - lndex Medicus -
`Exec/plat MOdICI)
`
`purihcation and characterization ol extracellular poly-
`ammo oxidaae produced by Penicillium so. No. PO—t
`(35A 31226)
`.
`.
`.
`by Y, Kobayashl and K. Horikoshl (Saltama, Japan)
`Charge transfer
`In peptides.
`lntramolecular
`radical
`transtormetione Involving methionine. tryptophan and
`tyros-ne (BEA 31231)
`by WA. Pratz, F. Slebert. J. Butler. E.J. Land. A. Menez
`and T. Montenay-Gareatl’or (Frelburg. F.Fl.G.. Manches-
`ter. U.K. and Gil-sur- Yvette and Paris, France) ........
`Charge traneler between tryptophan and tyrosine in
`proteins (BSA 31233)
`by J. Butler, E.J. Land. WA. Prutz and AJ. Swallow
`(Manchester, UK. and Frelburg. FR. G.) .............
`Characterization ol
`the proteolytlc activity tirmly at-
`tached to yeast phoephoenolpyruvate carboxyklnase
`(EBA 31227)
`by l. Beck, M, Muller and H. Holzer (Munich and Frei-
`bulg, ERG.) ...................................
`Isolation and characterization ol giycopeptidea oi hu-
`man transcortln (EBA 31224)
`I.V. Met-
`by 0A. Strel‘Chyonok, G.V. Awakumov.
`veentseva, L.V. Akhrem and AA. Akhrem (Minsk.
`us 53.) ......................................
`Isolation and purification ot rat acute-phase rag-macro-
`globulin (EBA 31229)
`by DE. Panrucker and FL. Loracheider (Calgary.
`Canada) ......................................
`Radioimmunoassay of rat acute-phase a,-macroglobu-
`tin (SBA 31230)
`(Calgary.
`by DE. Panrucker and EL. Lorachelder
`Canada) ......................................
`Puntication and partial characterization at two forms at
`urinary trypsin inhibitor (BBA 31235)
`by Y Tanaka. S. Maehara, H. Suml. N. Toki. S. Moriyama
`and K. Sasaki
`(Tokyo. Shimane. Mlyazalrl and
`Hiroshima. Japan) ..............................
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`The eltect ol aging on rat liver phosphoglycerate kinaale
`and comparison with the muscle enzyme (SBA 31237)
`by L.S. leemath and M. Rothatein (Buffalo. NY, U. 3.1!.)
`
`133
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`Purification and properties 01 the pyruvate dehydro-
`genaae complex from Salmonella typhimurlum and lor-
`mation ot hybrid: with the enzyme complex lrom
`Escherlchla coll (EBA 31239)
`by Fl. Seckler. R. Binder and H. Bleawanger (Tobingen.
`ERG.) .......................................
`Photoalllnity labeling ol membrane-bound porcine
`aminopeptideee N (BEA 31250)
`by D. Gralecoe. L. Varesl, M. Knrblehler and M. semerlva
`1 3a_/(Marselllee. France) .............................
`Demonstration that bovine erythrocyte cytochrome OS
`in the hydrophilic segment ol liver mlctosomal cyto-
`chrome b5 (BSA 31240)
`1V by SE. Slaughter. C.H. Wllllams, Jr. and D. E. Hultquial
`(Ann Arbor. MI, U.S.A.) ..........................
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`Ettect ot vanadate on the formation and stability 01 the
`phoephoenzyme terms 01 2.3-bisphoephoglycerate—dt
`pendent phosphoglycerate mutase and at phoe-
`phoglucomutne (EBA 312‘s)
`by J. Canerea, F. Climent. R. Ber-trons and G. Pan:
`(Barcelona. Spain) ..............................
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`The iaoelectrlc focusing at human thyroglobulln (EBA
`31236)
`by C. Davoh‘. s. Grimaldl, G. Rueca. M. Andrea/i and H.
`Edelhoch (Rome, Italy) ..........................
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`Isolation and partial characterization ol prochymosin
`and chymoein lrom cat (BEA 31244)
`by T.
`Jensen. N.H. Axelsen and B. Foltmann
`(Copenhagen, Denmark) .........................
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`Recombination kinetics lollowing nanosecond laser
`photolysis of carbonmonoxyhaemoglobln (SBA 31238)
`by R. Catterall. DA. Duddell, Fl.J. Morris and J.T.
`Richards (Salford, U. K.) ..........................
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`Circular dichroism studies on aa-antlplesmin and its
`interactions with pleemin and piaeminogan (BBA 31264)
`by T. Nilsaon,
`l. S/oholm and B. Wlman (Umea and
`Uppaala. Sweden) ..............................
`Purilication and characterization ol human Ct—esteraae
`inhibitor (BEA 31265)
`by T. Nileson and B. Wlman (Umoa. Sweden) .........
`
`163
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`137
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`17‘
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`' 134
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`192
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`200
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`210
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`21 a
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`22B
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`238
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`2‘3
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`2‘9
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`257
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`264
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`271
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`continued
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`INiIORMA'I'ION FOR CONTRIBUTORS TO BIOCHIMICA ET BIOPHYSICA ACT‘
`
`The 1932 edition 01 lhla booklet. otterlng detailed widen” to anthem le available tree 0! charge trom the IDA Edllorlel
`Secreterlet. P.O. Box 1345. 1000 EH Ameterdem. The Netherlende. It Ie eleo pubHehed ae Blochlm. Bloohya. Acta 715 (10.2) 1-23.
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`Page 3 of 10
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`

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`Tropomyofln Wucturo and Inhmobcular contacts In
`My paracryomln (BSA 31222)
`by 8.0. Gollldly (Chicago. IL. USA.) ..............
`
`277
`
`384 Reports
`
`Arthurian plot charmflstlco o! mombmno-bounq 3.
`hydvoxy-s-mothmluuryl cocnzymc A rating.» "om
`the mm of Hm brumomls (BBA 30018)
`by AB. Sipnt (saucer. Malayan) .................
`
`284
`
`Inhlblmry union of prollno-eonmnlng peptides on Xia-
`Pro—dlpomidytnminopoptidm (EBA 30023)
`by M. Honda, KM. Fukauwn. K. Fukulwl and T.
`Nagauu (Shiojin' and Yokohama, lean) ............
`Tides of "mod papon In other actions ............
`
`233
`291
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`Page 4 of 10
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`

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`Biochimica e! Biophysica Acid, 705 (1982) 2717276
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`Elsevier Biomedical Press
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`BEA 31265
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`271
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`PURIFICATION AND CHARACTERIZATION OF HUMAN CI-ESTERASE INHIBITOR
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`TORBJORN NILSSON and BJORN WIMAN ’
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`Department of Clinical Chemistry, Umed University Hospital, 5—901 85 Umet‘z (Sweden)
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`[Received December 16th, 1981)
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`Key words: Ci—eslerase.‘ Disulfide; Esterase inhibitor; Complement; (Human)
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`A new purification method for CI-esterase inhibitor is described, which is essentially a three-step procedure:
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`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
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`of about 100000 and NHz-terminal asparagine. The molecule is fully active as judged by complex formation
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`with CIs. Two of its three disulphide bridges can be easily reduced and S -carboxymethylated under
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`non-denaturing conditions without loss of activity. However, at high dithioerythritol concentration the third
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`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.
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`Introduction
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`Human plasma contains several protease inhibi-
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`tors of well-known functions. Among these,
`the
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`
`
`
`
`CI-esterase inhibitor is important in the regulation
`of the complement system [1] and possibly also in
`
`
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`
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`the kininogen system [2] and the intrinsic fibrinol-
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`
`
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`ysis [3]. Several purification methods for the CI-
`esterase inhibitor have been described [1,4—8], al-
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`
`
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`though few seem to give fully satisfactory prepara-
`tions suitable for functional studies. In order to
`
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`perform such studies we have therefore developed
`
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`a simple method for purification of CI-esterase‘
`inhibitor which can be operated on a large scale
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`and results in a high yield of pure product, with
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`
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`properties as close to the native molecule as possi-
`ble.
`
`
`Materials and Methods
`
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`Human plasma. Freshly frozen citrated human
`
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`plasma was obtained from the Blood Centre of
`
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`‘ To whom correspondence should be addressed.
`
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`0|67—4838/82/0000—0000/$02.75 ‘0 I982 Elsevier Biomedical Press
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`Page 5 of 10
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`Umea University Hospital (courtesy of Dr. B.
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`Cedergren).
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`C1_s. This was prepared essentially according to
`
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`
`
`the method of Chapuis et al. [9]. The final product
`
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`
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`was a homogeneous protein as established by
`SDS-polyacrylamide gel electrophoresis, all of
`
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`which could form a stable complex with Cl-
`esterase inhibitor, if this was added in excess.
`
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`Reagents. Hexyl-Sepharose was obtained by
`
`
`
`
`
`
`coupling 100 ml
`1 M hexylamine (Kebo AB,
`
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`
`
`Stockholm, Sweden), adjusted to pH 9 with hydro-
`
`
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`
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`chloric acid, to 1000 ml CNBr—activated Sepharose
`
`
`
`
`
`
`4B [10]. Sepharose 4B“ and DEAE-Sephacel“ were
`from Pharmacia Fine Chemicals
`(Uppsala,
`
`
`
`
`Sweden). Polybrene was from Aldrich (Beerse, Bel-
`
`
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`
`gium) and the chromogenic tri-peptide-p-nitroani—
`
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`
`
`lide substrate 5-2400 was a gift from Kabi peptide
`Research (Molndal, Sweden, courtesy of Dr. Lars
`
`
`
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`
`
`
`Mellstam). All reagents and chemicals used were
`
`
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`
`of analytical grade.
`
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`NH2-terminal amino acid determination. This was
`
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`performed by manual Edman degradation [1 l],
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`and the phenylthiohydantoins were identified by
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`thin-layer chromatography as described previously
`[12].
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`Page 5 of 10
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`

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`Amino acid analysis. The samples were hydro-
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`lyzed in constant boiling HC] in vacuo for 24, 48
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`and 72 h. Analysis was performed on an LKB
`
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`amino acid analyzer according to the procedures
`
`
`
`
`given by the manufacturers.
`Immunochemica.’ analysis. Rabbit antiserum to
`
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`the Cl-esterase inhibitor was raised by injection of
`
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`the purified protein mixed with Freund’s complete
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`adjuvant. Booster doses were given together with
`
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`Freund’s incomplete adjuvant when the specific
`
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`antibody titers were decreasing.
`Electroimmunoassay was performed according
`
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`
`to the method of Laurel] [13] and crossed im»
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`munoelectrophoresis essentially as described by
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`Clarke and Freeman [14}.
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`Determination of Cl} and CIT-esterase inhibitor
`concentrations. The C15 concentration was de-
`
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`termined from its absorbance at 280 nm, using an
`
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`Ago“ of_9.5 {15].
`The Cls activity was measured using the chro-
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`mogenic substrate 3-2400: a 140 nM solution of
`
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`Cls gave a AA“u of 0.043/mjn at a substrate
`
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`concentration of 0.63 mM in 0.] M sodium phos-
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`
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`phate buffer, pH 7.3, at 25°C.
`Cl-esterase inhibitor concentrations were de-
`
`
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`
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`termined spectrophotometrically using an Afioflm
`
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`
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`of 5.0 [11, or by titration against purified C15 in
`
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`the following way: 25 ,ul of a Cl-esterase inhibitor
`
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`sample was incubated for 10 min with 25 it] Cls
`
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`(final concentration 140 nM) in a cuvette. Then
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`0.7 m10.1 M phosphate buffer, pH 7.3, and 200 pl
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`3 mM 8-2400 were added and residual Cls activity
`
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`
`
`
`was measured from the AA4,0nm, as described
`
`above. The Cl-esterase inhibitor could then be
`
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`calculated from the decrease in AAMOnn-i'
`
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`Polyactylamide gel electrophoresis. SDS-poly-
`
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`
`
`acrylamide gel electrophoresis according to the
`
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`
`method 0; Weber and Osborn [16] was performed
`
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`on non-reduced and reduced samples, as previ-
`ously described [17].
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`Reduction and S-carboxymeihylation. Reduction
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`of the CT-esterase inhibito: was performed using a
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`protein concentration of about 2 mg/ml. Di-
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`thioerythritol was added to final concentrations of
`
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`003—200 mM. For reductions at non-denaturating
`
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`conditions, a 0.1 M sodium phosphate buffer, pH
`7.3, was used. For reductions at denaturing condi-
`
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`tions, the same buffer, containing 6 M guanidinium
`chloride, was used. After incubation for 30 min at'
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`272
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`Page 6 of 10
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`25°C in nitrogen athmosphere, [3H]ICH2COONa
`was added to a final concentration of 80 mM, and
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`
`incubation was continued in the dark for another
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`5 min. To the 200 mM dithioerythritol sample, 450
`
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`mM ICH2COONa was used. The labelled protein
`
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`was isolated by gel filtration on Sephadex (3-50
`
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`columns (0.8 cm2 X 9cm) equilibrated with 0.1 M
`
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`sodium phosphate buffer, pH 7.3. Control samples
`similarly treated,
`except
`that no di-
`were
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`
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`thioerythritol was added. The separated protein
`samples were analyzed by radioactivity measure-
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`
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`ment (Packard Tri-carb liquid scintillator) and
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`protein determination by AM or amino acid anal-
`
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`ysis after acid hydrolysis. As a reference protein,
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`az-antiplasmin (10 mg/ml) was treated in the same
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`way in order to obtain the specific radioactivity of
`the iodoacetic acid, since it
`is known that
`this
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`
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`protein contains three disulphide bridges and no
`
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`free SH group [18,19].
`
`
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`
`
`Results
`
`
`
`
`
`Purification of C1—-esterase inhibitor
`
`
`
`
`
`All procedure were carried out at +5°C. About
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`1 litre of plasma depleted in plasminogen by treat-
`
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`
`
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`ment with lysine-Sepharose [20] was thawed, made
`20 mM in EDTA, and poiybrene was added to a
`
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`final concentration of 0.6 g/l. The plasma was
`
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`subsequently diluted with an edual volume of PEG
`
`
`
`
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`
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`6000 (12%, w/v). After stirring for 30 min,
`the
`
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`precipitate was removed by centrifugation, and to
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`the clear supernatant were added about 300 ml of
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`DEAE-Sephacel”, equilibrated with 0.04 M phos-
`phate buffer, pH 7.0.
`
`
`
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`
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`After stirring for 30 min, the gel was washed on
`a Buchner funnel with about 15 litres of the same
`
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`phosphate buffer. The gel was then degassed and
`
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`packed in a column (5 X 15 cm), and subsequently
`
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`
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`eluted employing a linear gradient of NaCl from (J
`to 0.3 M (total volume 1800 ml) in the same phos-
`
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`
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`
`
`
`phate buffer (Fig. l).
`The Cl—esterase inhibitor concentration in the
`
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`
`
`eluted fractions was determined by electroim-
`
`
`
`
`
`
`
`munoassay. Generally it was eluted in the 0.15-
`
`
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`
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`0.18 M NaCl range. The fractions containing anti—
`
`
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`
`
`
`gen were pooled, concentrated to about 30 ml and
`made 0.4M in (NH4 )2504 by addition of an ap-
`
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`propriate amount of the solid substance.
`
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`The concentrated DEAE-Sephacel“ pool was
`
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`Page 6 of 10
`
`

`

`273
`
`esterase inhibitor were obtained from 1
`
`litre
`
`plasma.
`
`Characterization of the purified CT—esterase inhibi-
`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-
`duced and non-reduced samples (Fig. 3). 0n com-
`parison with reference proteins its molecular weight
`was estimated to about 100000. On crossed im-
`
`munoelectrophoresis, using a rabbit antiserum
`raised against the purified protein, only one pre-
`cipitation arc is observed for the purified Cl-
`csterase 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 Cl-esterase
`inhibitor in plasma. No signs of this inhibitor in
`complex with proteases such as Cls or Clr have
`been detected in normal plasma samples.
`The amino acid composition (Table I) is in ex-
`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
`[I] is, however, quite different. On Nth-terminal
`amino acid analysis, about lmol asparagine per
`mol protein was found. No other amino acid could
`be detected.
`
`Activity. Titration of the purified Cl-estcrase
`inhibitor with Cls resulted in an inverse linear
`relationship between the amount of Cl-esterase
`inhibitor added and remaining Cls activity. A 10%
`molar excess of the inhibitor was sufficient
`to
`
`abolish the Cls activity.
`SDS-polyacrylamide gel electrophoretic analy-
`sis of the reaction mixture obtained using Cls in
`
`'°
`
`b
`
`I”?
`
`I
`
`the
`Fig 3. SDS—polyacrylamide gel electrophoretograms of
`purified Cl-esterase inhibitor: (in), reduced sample: (b). non-re-
`duced sample.
`
`Absorbonceat280nm
`
`
`
`CNconcentrationlug/ml)
`
`Eluhon volume (ml)
`
`Fig. l. Elution of the DFAE-Sephacel column by a linear
`
`gradient from 0.04 M sodium phosphate buffer. pH 7.0, to the
`same buffer containing 0.3 NaCl. .4,“
`); Cl-esterase
`inhibitor (ClN) concentration according to Laurel] electro-
`phoresis (------).
`
`then applied to a column of hexyl-Sepharose (5 X
`20 cm), equilibrated with 0.4M (Ni-{02804 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 CT-esterase in-
`hibitor was pooled as indicated. and subsequently
`dialyzed against 0.1M sodium phosphate buffer,
`pH 7.3, in the cold. The solution was thereafter
`concentrated to give a final concentration of about
`3mg protein/ml and stored frozen at —80°C.
`Typically about 80—100 mg of the purified Cl-
`
`01.
`
`Absorbanceat280nm 9 N
`
`.O..
`
`I—O
`
`/_i__i___n.__i_
`MWWW'INBN.
`Elution volume (ml)
`
`Fig 2. Hydrophobic interaction chromatography on hexyl-Sep-
`harose. The pooled fraction from the DFAE-Sephacel column
`was applied to the hayl-Sepharoee column equilibnted with
`0.04 M sodium phosphate buffer containing 0.4 M (NH.),SO..
`The first peak consists of essentially pure Cl-eeterase inhibitor.
`
`Page 7 of 10
`
`

`

`274
`
`
`
`i-
`
`Fig. 4. Crossed immunoelectrophoresis of (a) purified CI-
`esterase inhibitor, (1)) normal human plasma. The second di-
`mension was run into a gel containing I% (v/v) rabbit anti-
`CT-esterase inhibitor.
`
`excess revealed that all of the CI-esterase inhibitor
`reacted to form a complex with the C75 light
`chain. When the Cl-esterase inhibitor was in ex-
`cess all of the Cls was converted to the enzyme-in-
`hibitor complex, indicating that both the inhibitor
`and the Cls preparations fully retain their activi-
`ties.
`
`Stability. A fresh preparation of Cl-esterase
`inhibitor, dialyzed against 0.1 M phosphate buffer,
`pH 7.3, was divided into aliquots and kept at
`+5°C, -20°C or —80°C. 'Sodiurn azide (0.04%)
`was added as preservant in the +5°C aliquots. At
`different times, ranging between 0—50 days, sam-
`ples were checked for inhibitory activity against
`Cls. 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
`
`Page 8 of 10
`
`TABLE I
`
`AMINO ACID COMPOSITION OF THE PURIFIED PRE-
`PARATION OF CI-FSTERASE INHIBITOR
`
`The figures represent the mean value of three determinations.
`
`
`
`
` Amino acid 1‘30“; Amino acid 1‘50“;
`
`Aspartic acid
`56.5
`Methionine
`14.5
`Threonine ‘
`59.9
`Isoleucine
`l9.8
`Set-inc '
`52.5
`Leucine
`62.7
`Glutamic acid
`61.0
`Tyrosine
`8.3
`Proline
`42.6
`Phenylalanine
`29.2
`Glycine
`17.0
`Histidine
`9.0
`Alanine
`38.0
`Lysine
`36.9
`Cysteine '
`5.5
`Arginine
`16.0
`Valine
`37.1
`
`‘ 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 I),
`thus forming three dis-
`ulphide bridges. This has been investigated further
`by reduction of the CI-esterase inhibitor with di-
`thioerythritol in the presence of 6M guanidinium
`chloride, followed by S—carboxymethylation with
`[3H1CH2COONa. Using az-antiplasmin as
`a
`standard, known to possess
`three disulphide
`bridges [19], we found 5.9 cysteines in the CI-
`esterase inhibitor. The small radioactivity incorpo-
`rated nonspecifically prior to reduction, corre-
`sponding to about 0.8 residues,
`is then already
`subtracted. Conclusively, the CI-esterase inhibitor
`contains three disulphide bridges.
`Partial reduction of the CI-esterase inhibitor
`under non-denaturing conditions using different
`dithioerythritol
`concentrations
`followed
`by
`S-carboxymethylation with [3H]ICH2COONa is
`shown in Fig 5. Two of the three disulphide bridges
`seem to be easily cleaved even at 3mM di-
`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 Cl-
`esterase
`inhibitor
`samples. Reduction and
`Scarboxymethylation of the two easily reduced
`disulphide bridges are performed without loss of
`
`

`

`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 az-antiplasmin could
`be reduced and S-carboxymethylated under non-
`denaturating conditions without affecting the
`inhibitory activity. Cleavage of
`the third dis-
`ulphide bridge could only be performed in the
`presence of denaturing agents and was accompa-
`nied by complete loss of activity [19,21]. In the
`present study, similar results are obtained with
`CI-esterase inhibitor, although the third dis-
`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-
`portant in maintaining the crucial conformation
`around the reactive site of the inhibitor. The simi-
`
`lar behaviour of the disulphide bridges in ail-anti-
`plasmin and Cl-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-
`jects l79/80 and 206/81). The expert technical
`assistance of Ms. Asa Alrnquist and Mr. Tord
`Arvidsson is gratefully acknowledged.
`
`References
`
`I Harpel, P.C. (1976) Methods FJIzymol. 45, 751-760
`2 Gigli, I., Mason, J.W., Colman, R.W. and Austen, K.F.
`(1970)}. lmmunoL 104. 574—58!
`3 Kluft, C. (1977) Haemoataaia 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, 0.1., Sim, RB. and Colomb, M.G.
`(1977) FEBS Lett. 79, 45-50
`6 Harpel, RC. and Cooper, N.‘P. (1975) J. Clin. Invest. 55.
`593-604
`7 Nagati, K., Iida. K. and Inai. S. (1974) Int. Arch. Allergy
`46, 935-908
`8 Anderson, W.H.K., Smith, J.K. and Fothergill, 1.5. (1975)
`Biochem. Soc. Trans. 3. 933-934
`
`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 Cl-esterase inhibitor are tedious
`and result in low yields and varying amounts of
`degraded forms of the inhibitor. The method re-
`cently published by Reboul et a]. [5] is simple, but
`in our hands it only gave a 50%-pure material with
`several contaminants at low concentrations. There-
`fore the present method seems to be superior in its
`simplicity, high yield and quality of the final prod-
`uct. A yield of 80—l00 mg/litre plasma represents
`about 45—55%, assuming a concentration of 180
`mg/l in normal plasma [1]. The present method
`can, furthermore, easily be operated on a large
`scale.
`
`The purified protein is a single-chain glycopro-
`tein with a molecular weight of about 100000, and
`asparagine is found to be its NHz-terminal amino
`acid. The purified preparation has electrophoretic
`mobility in the at2 region identical to that of the
`Cl-esterase inhibitor in plasma, as judged by
`crossed immunoelectrophoresis. It is also fully ac-
`tive, as judged by titration against Cls or analysis
`of a mixture of CI-esterase inhibitor and CIs in
`molar excess by SDS-polyacrylamide gel electro
`
`I‘M
`
`
`
`
`
`mot'SH/mot(IN u
`
`01
`
`onattivity
`
`10
`If)
`01‘! [manlrmion (mm
`
`m
`
`1W0
`
`Fig. 5. Reduction of Cl-esterase inhibitor (CIN) by different
`concentrations of dithioerythritol
`(DTE)
`followed
`by
`S-carboxylnethylation with [JHIICH ICOONa. The number of
`
`S-carboxymethylcysteinee were calcinated from the specific
`radioactivity (O
`O). Included in the figure are also the
`results from activity measurements (0).
`
`Page 9 of 10
`
`l l I l l l l l
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`

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