Chemical reagents were designed to cross-link and connect hemoglobin and superoxide dismutase, combining the oxygen transport and superoxide-removal capabilities of the red cell in a dual-function protein. Reaction of 1 with thiol-protected hemoglobin followed by reduction produces cross-linked hemoglobin with a free thiol on the cross-link. Reaction of SOD with 5 produces a cross-linked protein with a maleimide on the cross-link. Addition of the hemoglobin-thiol to the SOD-maleimide produces a protein with the desired dual properties. Hemoglobin's oxygenation cooperativity is lowered as a result of being in the conjugate, while SOD's activity is equal to that of the native protein.

Bis-Tetramers of Hemoglobin Containing Disulfide Linkages Mlaster of Science, 2000, Amer Alagic Department of Chemistry, University of Toronto Hemoglobin tetramers cross-linkeû and wmected to a second hemoglobin molecule (bis-tetramers) are a new c h of oqgens4nyyI8 proteins with interesthg properties. Hemoglobin bis-tetramers f o n d fiom the teaction ofhemogiobin with tetdmctional ratgents bind oxygen without cooperative fatufes found in tetramers. This indicates that the dynamics of individuai hemogîobins within bis-tetramers are dEerent thui those of native or cross-ünked hemoglobin mono-tetramers. This thesis reports the synthesis ofa new t*nfunctionai cross-linking reagent 1 and its reaction with human hemoglobin A The reagent contains a disuffide bond that diows for the cleavage and refomtion of the ünk between tetramers. 1 modifies hemogiobin in a sitespecitic manna, cross-linking the amino groups of Val4 and Lys82 residues of the B giobin chains* resulting in the formation of hemoglobin bis-tetramers thst cm be cleaved at the disulnde bond by reduaion. The mamurement of the physicai propaties of these unique bis-tetramers and of the resulting crodinked hemoglobii tetramers, which are fonned by cleavage ofthe hemoglobin bis-tetramers, will provide a cornparison of the bisand monoStates. Thew metsunmaits am cxpecttd to give insight hto the dynamics ofhcmoglobin molecules within the bis-tetramers and to cornlate biochemkal properties and stnicture. Potentiai uses of 1 dso iaclude formation ofhmogiobih-cnsyme conjugites with novd intemain8 properties. 1 would üke to t lwk Professor Ronald Kluger for giving me the opportunity to work on this intaesthg project, and for his help and guidance throughout. I would dso iike to thank members ofour group for their help and advice: Dr. John Pezaclri, Steven Brookes, Chung-Woo Fung, Vittorio De S t b , L i a Cameron, Ian Moore, Ayesha Saîih, Peter Spencer, Anna Teytelboym, Jie Zhang, and Daria Yu. 1 would Mce to thank to my family for their help and support throughout my studies. Bis-Tris CD Da DBIT DPEE DPG DTT EDTA ESI(MS) FPLC Hb HPLC MOPS m.p. SEC SDS-PAGE TMMP TTDS bis(2-hydtoxyethy1)zUninotns(bydroxymethyl)m~me circuiar dichroism Daltons N,N' -5, S ' b i s [ b i s ( 3 , S & % r o m o s a i i c y l ) i w p h 1.2-Bis(2-[3 ,Sbis(3~5~brornodsyidonly)phenoxy]ethoxy~e 2.3-diphosphogiycerate dithiothreitol ethelenediahetetraacetic acid electrospray ionization rnass spectroscopy f~ protein liquid chromatography hemoglobin high pressure liquid chromatography 3-(N-morpholino)propanesulfonic acid melting point size exclusion chromatography sodium dodecyl aiifhte-polyacrylamide gel electrophoresis trimesoyltris(methyl phosphate) trimesoyltris(3,5-diaromosalicylate) TABLE OF CONTENTS .. ............................................................................................. ARSTRACT. ........ . II ... .......................................................................... Ackmnvledgnent s. .... ......*.............. I I I . . List of Abbrmatrons ....................*...... ............................ .....*~..~................................ N CHAPTER I= INTRODUCTION ................................................................................. 1 ................................ .....*....*......................*...............................***.. 21. Materials ,., 10 ..................................................................... 2.2 S'ihesis of c~osp-IinRing reagent 11 2.2.1. Synthesis of 4,4'.dithiobisbenzoic acid, la ................................................. 11 ......................................................................................... 2.2.2. Synthesis of 1 b 12 2.3. Cros~hhng reaction of huma hernogiobin A with 1 ..................................... 15 2.3.1 . Reaction of cross-linked hemogiobin produas with DTT ........................... 15 2.3.2. Reaction ofDTT-treated modifieâ hemogiobin products with azodicarôoxyiic acid bisdimethylamide, (Diamide) ....................................................................... 16 2.4. A m &sis of m d p e d kmogiobim ................................................................... 16 2.4.1 . Chromatography ........................................................................................ 16 2.4.2. SDS-PAGE malysis .................................................................................. 17 2.4.3. Enzymatic hydrolysis of unmodifieci and modifiai B gîobin chahs .............. 18 .................... 3.1. Sjnthesis of cross-linking reagent .. ..................................... 2 0 3.2. Crosshbng reaction of himan hcmogiobin A wifh 1 and unuiysis of the reaction prtxhcts* ................................................................................................... 21 3.2.1. Chromatognphy ....................... .. ........................................................ 21 3.2.2. SDS-PAGE analysis .......................... . . . . 33fsobtian d chvrractm*&üion of hemogiobin biste fnrniers .....CC...C................. -29 3.4. Ehymanc Iiycitidls of u)MIoI.ljed <Pdmd@eà f l giobiii cWm ................ ... 31 3.5. W spctraijwoperllres of kmog/ob& bis-tehainors ................. . ................ 32 4.1. Amlysis of the nrdped heinoglobins fmed U n g the reuctim of hm011 .............................................................................................. hemogllobm A with 1 3 5 4.2. Silggesttsnons for Fume WorA ........................................................................ 4 2 CONCLUSIONS ......................................................................................................... 46