= urnal
`Europ
`of Biochemistry
`
`Editorial Board
`Honorary Chairman : Sir Hans Krebs (Oxford)
`Editor-in -Chief: Claude Liebecq (Liege)
`J. leuan Harris (Cambridge), Lothar Jaenicke (Koln) ,
`Associate Ed i tors: Giorgio Bernardi (Paris) ,
`Cees Veeger (Wageningen)
`Edi tor s: F. Franek (Praha) , G. P. Georgiev (Moskva), H. G . Hers (Bruxelles) ,
`T. Keleti (Budapest), M. Lazdunski (Nice) , P. Overath (Tubingen),
`J. G. Re ich (Berlin) , M. Revel (Rehovot), M . Sela (Rehovot), C . A. Vernon (London) ,
`J.-P. Waller (Palaiseau), D . H. Williamson (Oxford), H. G. Zachau (Munchen) ,
`W . Z il lig (Martinsried)
`Special Advisor to the Editor-in-Ch ief : H. Mayer-Kaupp (Heidelberg)
`Advisory Board
`R. Banerjee (Paris) , E. K . F. Bautz (Heidelberg) , H. Beaufay (Bruxelles) ,
`C . Bagl ioni (Albany),
`L. D . Bergelson (Moskva) , L. Boross (Szeged), F. Bossa (Camerino) ,
`E. M . Bradbury (Portsmouth), R. Braun (Bern), H. Bue (Paris), P. H. W . Butterworth (London),
`E. Carafoli (Zurich) , M . J. Clemens (London), Sir John Cornforth (Brighton),
`K. Decker (Freiburg i. Br.), G . H. de Haas (Utrecht), W . Doerfler (Koln) , J.-P. Ebel (Strasbourg) ,
`W . C. Fiers (Gent) , C . Gancedo (Madrid), P. B. Garland (Dundee) , J.-M . Ghuysen (Liege) ,
`D . B. Gowe r (London), D. 0 . Ha ll (London) , E. J.M . Helmreich (Wurzburg) ,
`P. Hemmerich (Konstanz) , B. Hirt (Lausanne), E. Hofmann (Leipzig) ,
`0 . Hoffmann-Ostenhof (Wien) , R. Jaenicke (Regensburg) , P. Jolles (Paris),
`M . Ya . Karpeisky (Moskva) , E. Katchalski-Katzir (Rehovot) , K. Kirschner (Basel) ,
`M . Klingenberg (Munchen), H. Klostermeyer (Kiel), G. L. E. Koch (Cambridge) ,
`L. Kovac (lvanka pri Dunaji) , C. G. Kurland (Uppsala) , U. G. Lagerkvist (Goteborg) ,
`A. Levitzki (Jerusalem) , U. Z . Littauer (Rehovot) , R. Lontie (Leuven) ,
`0 . Luderitz (Freiburg i. Br.) , S . Magnusson (Aarhus) , C . Milstein (Cambridge) ,
`R. Monier (Villeju if), K. Mosbach (Lund) , E. A. Newsholme (Oxford) , D . Oesterhelt (Wurzburg) ,
`S . Orrenius (Stockholm), T. A. J. Payens (Ede) , D. W . G. Pette (Konstanz),
`R. J. Planta (Amsterdam) , J. Ricard (Marseille) , R. Rigler (Stockholm) ,
`A. Schellenberger (Halle/ Saale), M . C. Scrutton (London), G. Semenza (Zurich) ,
`A . Sentenac (Saclay) , S . Shaltiel (Rehovot) , N. Sharon (Rehovot), D. Shugar (Warszawa),
`F. $orm (Praha), F. S . Steven (Manchester) , H. Sund (Konstanz), R. Thauer (Marburg/ Lahn),
`R. Thomas (Rhode-St-Genese), K. F. Tipton (Cambridge), A. Trebst (Bochum) ,
`D . R. Trentham (Philadelphia), R. Tsanev (Sofiya) , V. Ullrich (Homburg), K. van Dam (Amsterdam),
`D. Vazquez (Madrid) , L.-V. von Stedingk (Stockholm), V . P. Whittaker (Gottingen),
`0 . W ieland (Munchen), M . Wiewiorowski (Poznan), L. Wojtczak (Warszawa)
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 1 of 13
`
`

`

`European Journal of Biochemistry
`Volume 78 Number 1 August (II) 1977
`
`83
`
`Contents
`Repetitive and Non-repetitive Sequences in the Transcript in vitro of Porcine Thyroid Chromatin
`V. E. Avvedimento , A. M . Acquaviva , and S. Varrone
`11 Hydrophobic and Carbohydrate-Recognition Chromatographies of Collagen Glucosyltransferase
`H. Anttinen , R. Myllyla, and K. I. Kivirikk o
`19 Purification and Characteriza tion of Human Antiplasmin, the Fast-Acting Plasmin Inhibitor in Plasma
`B. Wiman and D . Collen
`27 The Isolation of Tubulin and Actin from Mouse 3T3 Cells Transformed by Simian Virus 40 (SV3T3 Cells) ,
`an Established Cell Line Growing in Culture
`K. Weber, R. Koch , W . Herzog, and J. Vandekerckhove
`33 Glycolipids of the Human Gastric Content. Structure of the Sulfated Glyceroglucolipid
`B. L. Slom iany,·A. Slomiany, and G. B. J. Glass
`41 Carbohydrate Incorporation in Plasma Membranes of Mouse Thymocytes Stimulated by Concanavalin A
`R. V . W . van Eijk and P. F. Muhlradt
`55 Specificity of Elongation Factor Tu from Escherichia coli with Respect to Attachment of the Amino Acid
`to the 2' or 3'-Hydroxyl Group of the Terminal Adenos in e of tRNA
`M . Sp rinzl, M. Kucharzewski , J. B. Hobbs, and F. Cramer
`63 Refinement of the Solution Confo rmation of Valinomycin with the Aid of Coupling Constants from the
`13 C-Nuclear- Magnetlc-Resonance Spectra
`V. F. Bystrov , Yu. D . Gavrilov, V. T. Ivanov, and Yu . A. Ovchinnikov
`Induction of D ivision Synchrony in Tetrah ym ena pyriformis by a Single Hypoxic Shock. Its Use in
`Elucidating Control of the Cell Cycle by Adenosine 3': 5'-Monophosphate
`J. R. Dickinson , M . G. Graves , and B. E. P. Swoboda
`89 The Incorporation of [ 14C]Glucosamine into Dol ichol Diphosphate N-Acetyl[ 14C]glucosam ine by Unbroken
`Liver Cells in Culture
`J. R. Cooper and F. W. Hemming
`95 Studies on Nucleotidases in Plants . Isolation and Properties of the Monomeric Form of the C rystalline and
`Homogeneous Mung Bean Nucleotide Pyrophosphatase
`C . V. Balakrishnan , C . S . Vaidyanathan, and N . A Rao
`103 Chorismate Mutase/ Prephenate Dehydratase from Escherichia coli K 12. Modification with 5,5' -Dithio(cid:173)
`brs(2-nitrobenzoic acid)
`M .-J. H. Gething and B. E. Davidson
`111 Chorismate Mutase/ Prephenate Dehydratase from Escherichia coli K12. Effects of Chemical Modification on
`the Enzymic Activities and Allosteric Inhibition
`M.-J. H. Gething and B. E. Davidson
`119 Purification , Crystallisation and Preliminary X-Ray Studies on Avian Pancreatic Polypeptide
`S. P. Wood , J. E. Pitts, T. L. Blundell , I. J. Tickle , and J. A. Jenkins
`127 The Interactive Binding of Two Ligands by an Allosteric Protein
`R. F. Steiner and L. Greer
`133 Structural Rearrangements Due to Ligand Binding and Haem Replacement in Myoglobin and Leghaemoglobins
`N. A. Nicola and S. J. Leach
`Isolation and Characterization of Two Methionine : tRNA Ligases from Wheat Germ
`M . D. Rosa and P. B. Sigler
`153 Physical Studies on the Conformati on of Ribosomal Protein S4 from Escherichia coli
`C. A. Morrison , R. A. Garrett, and E. M . Bradbury
`161 Demonstration of Two a-Globin Genes per Human Haploid Genome for Normals and Hb J Mexico
`P. Tolstoshev, R. Williamson, J. Eskdale , G. Verdier, J. Godet, V . Nigon, G. Trab uchet, and M. Benabadji
`(Continuation overleaf)
`
`141
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 2 of 13
`
`

`

`Contents (Continuation)
`
`183
`
`167 Microtubule Assembly in vitro. Purification of Assembly-Promoting Factors
`A. Fellous, J. Francon , A.-M . Lennon , and J. Nunez
`175 Reverse Transcription of Thyroglobulin 33-S mRNA
`C. Dinsart, F. van Voorthuizen , and G. Vassart
`Influence of 1,2,3-Benzene-tricarboxylate on Pyruvate Metabolism in Rat-Liver Mitochondria
`J. W . Stucki
`189 Phytohaemagglutinin Stimulation of Human Lymphocytes during Amino-Acid Deprivation. RNA Polymerase I
`Activity of Isolated Nuclei
`C. Dauphinais and W. I. Waithe
`195 The Size and Shape of Human and Bovine Antithrombin Ill
`B. Nordenman, C. Nystrom , and I. Bjork
`205 Electron Microscopy Analysis of the Interaction between Escherichia coli DNA-Dependent RNA Polymerase
`and the Replicative Form of Phage fd DNA. 1. Mapping of the Binding Sites
`P. U. Giacomoni, E. Delain, and J. B. Le Pecq
`215 Electron Microscopy Analysis of the Interaction between Escherichia coli DNA-Dependent RNA Polymeras
`and the Replicative Form of Phage fd DNA. 2. Analysis of the Dissociation Kinetics
`P. U. Giacomoni , E. Delain, and J. B. Le Pecq
`221 Prostatic Binding Protein. A Steroid-Binding Protein Secreted by Rat Prostate
`W. Heyns and P. de Moor
`231 Studies on the Stimulation by Ca 2+ and the Inhibition by ADP of Steroid 11 ~-Hydroxylation in Adrenal
`Mitochondria
`A. M. Moustafa and S. B. Koritz
`239 Modification of L-lsoleucyl-tRNA Synthetase with L-lsoleucyl-Bromomethyl Ketone . The Effect on the
`Catalytic Steps
`P. Rainey, B. Hamm er-Raber, M.-R . Kula, and E. Holler
`251 Studies on Protein-Phosph orylation Reactions in Isolated Chromatin
`J. Bohm, G. Keil, and R. Knippers
`267 Functional Roles of 50-S Ribosomal Prote ins
`F. Hernandez, D . Vazquez, and J. P. G. Ballesta
`273 Purification and Primary Structure of Cytochrome f from the Cyanobacterium, P/ectonema boryanum
`A. Aitken
`281 The Amino-Acid Sequence of Trout-Testis Histone H1
`A. R. Macleod , N. C. W . Wong , and G. H. Dixon
`293 Oxidation Kinetics of 1,5-Dihydroflavin by Oxygen in Non-aqueous Solvent
`V . Favaudon
`309 Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase . A Comparative Study of the
`Enzymes from Yeast and Sturgeon Muscle
`A. F. Chaffotte, C. Roucous, and F. Seydoux
`
`Indexed in Current Contents
`
`SEDVA FEINBIOCHEMICA
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`A4
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 3 of 13
`
`

`

`European Journal of Biochemistry
`
`This JOURNAL continues the tradition of "Biochem.ische Zeitschrift" founded in 1906 and edited by
`C.Neuberg (-Vol.246), C.Neuberg and W. Grassmann (-Vol.279), W .Grassmann (-Vol.317), F.G.Fischer
`and K.Lang (-Vol.325), and by a group of leading German biochemists unto the end. Upon completion of
`Vol.346 its publication was discontinued in favour of closer European cooperation.
`
`The EUROPEAN JOURNAL OF BIOCHEMISTRY will primarily publish papers on fundamental aspects of
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`or physical level, or that they describe new methods applicable to biochemical problems.
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`Manuscripts should conform to the "Instructions to Authors" (overleaf) and should be submitted, in
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`Copyright <C> by the Federation of European Biochemical Societies 1977 .
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 4 of 13
`
`

`

`Volume 78 1977
`
`f/E/B/8/
`European Journal
`of Biochemistry
`
`Editorial _Board
`Honorary Chairman : Sir Hans Krebs (Oxford)
`Editor-in-Chief: Claude Liebecq (Liege)
`Associate Editors: Giorgio Bernardi (Paris), J. leuan Harris (Cambridge), Lothar Jaenicke (Koln),
`Cees Veeger (Wageningen)
`Editors : F. Franek (Praha) , G. P. Georgiev (Moskva), H. G. Hers (Bruxelles),
`T. Keleti (Budapest), M. Lazdunski (Nice) P. Overath (Tubingen),
`J. G. Reich (Berlin), M. Revel (Rehovot), M. Sela (Rehovot), C. A. Vernon (London) ,
`D. H. Williamson (Oxford), H. G. Zachau (Munchen),
`W. Zillig (Martinsried)
`Special Advisor to the Editor-in -Chief : H. Mayer-Kaupp (Heidelberg)
`Advisory Board
`C. Baglioni (Albany), R. Banerjee (Paris), E. K. F. Bautz (Heidelberg) , H. Beaufay (Bruxelles),
`L. D. Bergelson (Moskva) , L. Boross (Szeged) , F. Bossa (Camerino) ,
`E. M . Bradbury (Portsmouth), R. Braun (Bern), H. Bue (Paris) , P.H. W . Butterworth (London),
`E. Carafoli (Zurich), M. J. Clemens (London) , Sir John Cornforth (Brighton) ,
`K. Decker (Freiburg i. Br.) , G. H. de Haas (Utrecht) , W . Doerfler (Koln) , J.-P. Ebel (Strasbourg),
`W . C . Fiers (Gent) , C . Gancedo (Madrid), P. B. Garland (Dundee) , J.-M . Ghuysen (Liege) ,
`D. B. Gower (London) , D. 0. Hall (London) , E. J. M . Helmreich (Wurzburg),
`P. Hemmerich (Konstanz) , B. Hirt (Lausanne), E. Hofmann (Leipzig),
`0 . Hoffmann-Ostenhof (Wien) , R. Jaenicke (Regensburg), P. Jolles (Paris),
`M . Ya . Karpeisky (Moskva), E. Katchalski-Katzir (Rehovot), K. Kirschner (Basel),
`M . Klingenberg (Munchen) , H. Klostermeyer (Kiel) , G . L. E. Koch , (Cambridge) ,
`L. Kovac (lvanka pri Dunaji) , C . G. Kurland (Uppsala) , U. G . Lagerkvist (Goteborg),
`A. Levitzki (Jerusalem), U. Z. Littauer (Rehovot), R. Lontie (Leuven) ,
`0 . Luderitz (Freiburg i. Br.), S. Magnusson (Aarhus), C . Milstein (Cambridge) ,
`R. Monier (Villejuif), K. Mosbach (Lund) , E. A. Newsholme (Oxford), D. Oesterhelt (Wurzburg),
`S. Orren ius (Stockholm) , T. A. J. Payens (Ede) , D. W . G. Pette (Konstanz) ,
`R. J. Planta (Amsterdam), J. Ricard (Marseille), R. Rigler (Stockholm),
`A. Schellenberger (Halle/ Saale), M . C . Scrutton (London), G. Semenza (Zurich),
`A. Sentenac (Saclay) , S. Shaltiel (Rehovot), N. Sharon (Rehovot), D . Shugar (Warszawa),
`F. $orm (Praha), F. S. Steven (Manchester) , H. Sund (Konstanz) , R. Thauer (Marburg/Lahn),
`R. Thomas (Rhode-St-Genese), K. F. Tipton (Dubl in), A. Trebst (Bochum),
`D . R. Trentham (Philadelphia) , R. Tsanev (Sofiya), V. Ullrich (Homburg), K. van Dam (Amsterdam),
`D. Vazquez (Madrid), L.-V. von Stedingk (Stockholm), V . P. Wh ittaker (Gottingen),
`0 . Wieland (Munchen), M. Wiewiorowski (Poznan) , L. Wojtczak (Warszawa)
`
`Published by Springer-Verlag Berlin Heidelberg New York
`on behalf of the Federation of European Biochemical Societies
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 5 of 13
`
`

`

`Eur.J. Biochem . 78, 133 - 140(1977)
`
`Structural Rearrangements Due to Ligand Binding and Haem
`Replacement in Myoglobin and Leghaemoglobins
`
`Nico.s A. NICOLA and Sydney J. LEACH
`
`Department of Biochemistry, •University of Melbourne
`
`(Received July 30, 1976/June 1, 1977)
`
`Structural rearrangements in sperm whale myoglobin and leghaemoglobins caused by changes in
`the spin or oxidation state of the iron as a consequence of ligand binding have been measured by
`difference spectroscopy in the ultraviolet. When compared with the high-spin acetate complex,
`ligands which cause a transition to the low-spin state also cause large perturbations of tyrosine(s)
`remote from the haem pocket in myoglobin but only minor perturbations of tryptophan(s) in legha(cid:173)
`emoglobin. This may indicate a weaker coupling between events at the haem site and conformational
`changes in the protein in leghaemoglobins. The absorption spectra of various haem-liganded forms
`of the two proteins as well as the binding of the dye rose Bengal to the two apoproteins are consistent
`with weaker interactions between the haem and apoprotein and a more solvent-exposed haem pocket
`in leghaemoglobin compared with myoglobin.
`
`Leghaemoglobins are plant haemoproteins which
`resemble animal myoglobins with respect to their
`molecular weight, monomeric state and their ligand(cid:173)
`binding (especially oxygen-binding) properties. Their
`high oxygen affinity is presumed to be central to their
`biological function in the root nodules of legumes (see
`e.g. [1 ]). Most of the reported structural, spectroscopic
`and ligand-binding studies have been on the major
`leghaemoglobin component from soybean (Glycine
`max, er Lincoln). It has recently been shown that this
`plant produces several leghaemoglobins of differing
`amino acid sequence but similar overall conformation
`[1 , 2], that these characteristics are probably general
`in Leguminosae (Thul born et al., unpublished) and that
`the most readily discemable differences between
`leghaemoglobins of widely different origin and amino
`acid sequence, is their antigenicity [3]. Comparing
`the leghaemoglobins with sperm whale myoglobin
`reveals interesting similarities and differences [2, 4].
`For the purposes of the present paper, the most
`pertinent conclusion was that the haem moiety may
`be more loosely held in the leghaemoglobins than in
`myoglobin and there is less resistance to changes in
`spin state on ligand binding in the former case. This
`is consistent with the observations that soybean
`leghaemoglobin binds nicotinic acid with much higher
`affinity [5, 6] and its haem moiety is more readily
`exchangeable [7] than in myoglobin. Evidence has
`
`Abbreviation. CD, circular dichroism.
`
`also been adduced that the haem pocket in the plant
`globin is not as non-polar as that in myoglobin [8 , 9] .
`The purpose of the studies described here was to
`seek evidence of structural changes occurring in both
`types of haem proteins arising from changes in spin
`state. The reason for our interest in such changes is
`the observation by Perutz [10] that high-spin to low(cid:173)
`spin transitions in tetrameric haemoglobin are ac(cid:173)
`companied by deep-seated molecular rearrangements
`but little is known of corresponding changes in
`monomeric systems. In order to compare them with
`myoglobin and to generalise from the results, several
`leghaemoglobins and a variety of ligands (including
`nicotinate) and both oxidation states were used.
`Changes in absorption spectra in the visible, Soret and
`near-ultraviolet were measured, usually by the dif(cid:173)
`ference technique. Finally, the dye rose Bengal was
`used as a spectroscopic indicator of the polarity of the
`haem pockets in the two types of proteins.
`
`MATERIALS AND METHODS
`
`Materials
`
`The dye rose Bengal (3 ' 4' 5' 6'-tetrachloro-2,4, 5, 7-
`tetraiodofluorescein disodium salt) was obtained from
`Fluka. Chlorohemin was lot 8071 from Nutritional
`Biochemicals Corp. and was recrystallised according
`to Fischer [11]. Leghaemoglobins were extracted and
`purified as already described [2,4]. Sperm whale
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 6 of 13
`
`

`

`134
`
`Structural Effects of Liganding in Myoglobin and Leghaemoglobins
`
`myoglobin was batch 10 from Seravac or batch 52348
`from Koch-Light.
`Potassium ferricyanide, potassium cyanide, potas(cid:173)
`sium cyanate, sodium acetate, sodium azide, sodium
`dithionite and sodium fluoride were AR grade from
`British Drug Houses. Nicotinic acid was AR grade
`from Townson and Mercer and imidazole from Sigma.
`Other chemicals were AR grade and glass-distilled
`water was used throughout.
`
`Concentration Estimations
`
`Absorption spectra for
`the reduced pyridine
`haemochromes of soybean, serradella and lupin leg(cid:173)
`haemoglobins were identical with those for sperm
`whale myoglobin. Since the spectra for other com(cid:173)
`plexes of these proteins were much more variable than
`this, the formation of pyridine haemochromes was
`used as a measure of haemoprotein concentration as
`in previous studies [2, 4].
`
`Formation of Liganded Components
`
`Most liganded complexes of leghaemoglobin and
`myoglobin were formed simply by titration with the
`appropriate ligand until no further spectral changes
`could be observed. The deoxyferrous and oxyferrous
`complexes were formed as described previously [4]
`and the percentage of oxyferrous and ferric forms was
`assayed as described in the same paper.
`
`Formation of Apoproteins
`
`Apoproteins of myoglobin and leghaemoglobins
`were made from the haem proteins by the acetone/HCI
`method of Rossi-Fanelli et al. [12]. Acetone (BDH)
`was redistilled from potassium permanganate and
`Drierite. Glass-distilled water was de-ionised using a
`Bio-Rad mixed-bed ion-exchange resin. If the removal
`was carried out at - 20 °C the products usually had
`less than 1 % of the original haem content as judged
`from the A400/ A 280 ratio and were recovered in about
`70 % yield. Their concentrations and those of the haem
`proteins were determined as previously described
`[2, 4) . Molar absorption coefficients at 280 nm in
`phosphate pH 7.5 were 17900 and 16300 M - 1 cm - 1
`for soybean apoleghaemoglobin and sperm whale
`apomyoglobin respectively.
`
`Absorption Spectra and Difference Spectra
`
`Absorption spectra were measured with a Cary 14
`spectrophotometer. Difference spectra were usually
`run on this instrument using conditions similar to
`those reported by Nicola and Leach [13) except that
`a four-cell arrangement was usually used to allow
`
`compensation for the addition of absorbing ligands
`to the sample cell.
`
`Circular Dichroic Spectra
`
`The instruments and procedures used for measur(cid:173)
`ing CD spectra were those described by Nicola et al.
`[4].
`
`RESULTS
`Effect of Spin and Oxidation State on Sorel and
`Visible Absorption Spectra
`
`Spectral data for soybean leghaemoglobin a and
`the main lupin component have been reported by a
`number of workers [1 ,9, 14-16) and these have been
`supplemented by data for the aquoferric, fluoroferr ic
`and nicotinoferric as well as the oxyferrous and deoxy(cid:173)
`ferrous soybean derivatives [4]. In the present work,
`Soret and visible absorption spectra have been
`measured for the acetate, cyanate, azide and imidazole
`derivatives of ferric soybean leghaemoglobin. All of
`these spectra are best discussed by grouping the deri(cid:173)
`vatives as follows: high-spin ferrous= deoxy; low(cid:173)
`spin ferrous= oxy, pyridine and nicotinate; high-spin
`ferric= acetate and fluoride; low-spin ferric= cyanide,
`imidazole and nicotinate; mixed-spin ferric=aqu o,
`cyanate, azide.
`The spectra for soybean leghaemoglobin a are very
`similar to those reported for lupin leghaemoglobin [9) .
`They are also quite similar in general form to those
`for sperm whale myoglobin [17) with corresponding
`complexes showing the same spin-state tendencies.
`However, there are differences in the peak absorption
`intensities and their wavelengths. Table 1 shows that
`the Soret band for all classes of leghaemoglobin
`spectra consistently occurs at shorter wavelengths
`(blue shift) than for corresponding complexes of
`myoglobin independent of the spin or oxidation state
`of the iron. Also the mixed-spin complexes for leg(cid:173)
`haemoglobin appear to be further towards the low(cid:173)
`spin state than those for myoglobin (not shown).
`
`Binding of Nicotinic Acid
`
`The binding of nicotinic acid with relatively high
`affinity to both ferrous and ferric leghaemoglobin is
`of some interest because this ligand does not bind to
`myoglobin except at much higher concentrations [18)
`and because it has been postulated that it may play a
`regulatory role in vivo. This hypothesis and the binding
`of nicotinic acid to soybean leghaemoglobin a has
`been discussed in detail by Appleby et al. [1,5,6). We
`have extended these observations to show that the
`high-affinity binding of nicotinic acid is probably a
`general phenomenon for leghaemoglobins. Thus, leg-
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 7 of 13
`
`

`

`Nicos A. Nicola and Sydney J. Leach
`
`haemoglobins from serradella, snake bean and lupin
`all showed changes in absorption spectra for their
`ferrous forms (reduced with sodium dithionite) when
`titrated with nicotinic acid in 0.01 M phosphate buffer,
`pH 7.0, which were qualitatively similar to those of
`the soybean protein [5,6]. The structural consequences
`of nicotinic acid binding are best seen using difference
`spectra. Fig. 1 shows such a set of spectra for the
`titration of ferric soybean leghaemoglobin a in the
`near-ultraviolet (Fig. 1 A) and visible (Fig. 1 B) regions.
`The visible difference spectra show the progressive
`decrease in the charge-transfer band intensities at 494
`and 629 nm and the increase in the ix and /J band
`intensities at 530 and 556 nm respectively. This is
`typical for a high-spin to low-spin transition.
`The changes which occur concomitantly in the
`near-ultraviolet, (Fig. 1 A), are perhaps more interest(cid:173)
`ing. These changes run parallel with those in the visible
`
`Table 1. Wavelengths of the main Sorel peak for various complexes
`of sperm whale myoglobin , soybean leghaemoglobin a and lupin
`leghaemoglobin a
`
`Complex type
`
`Ligand Wavelength for
`
`sperm whale soybean
`Mb [17]
`Lb
`
`lupin
`Lb [9]
`
`Low-spin ferric
`High-spin ferric
`Mixed-spin ferric
`Low-spin ferrous
`High-spin ferrous
`
`CN
`F
`H20
`02
`
`nm
`
`423
`406
`410
`41 8
`434
`
`0 .06
`
`417
`403
`403
`412
`427
`
`416
`403
`404
`
`421
`
`A
`
`135
`
`and, in fact, some of this difference absorption is due
`to spectral changes in the haem. Strickland et al. [19]
`however, have shown that neither the haem nor the
`haem undecapeptide of cytochrome c (which contains
`no aromatic amino acids) display any fine structure
`bands in the haem absorption region between 250 and
`310 nm, even at liquid nitrogen temperatures, so the
`haem cannot be the origin of the sharp difference
`peaks seen in Fig. 1 (A). The peaks below 280 nm may
`well arise from the transfer of nicotinic acid from an
`aqueous environment into the non-polar haem pocket,
`since the parent spectrum of nicotinic acid shows
`peaks at 270, 264 and 257 nm. However, the negative
`difference peak at 294 nm and probably the one at
`286 nm must arise from a change in the environment
`of a tryptophan residue and this change is proportional
`to the extent of binding of nicotinate. Such negative
`difference peaks, superimposed on an increasingly
`positive haem background, suggests that the trypto(cid:173)
`phan(s) become more exposed to aqueous solvent as
`the titration proceeds.
`
`'Form ' Difference Spectra in the Near-Ultraviolet
`'Form' difference spectra (i.e. the difference spectra
`for various liganded complexes against the pure high(cid:173)
`spin acetate complex) were recorded in the near-ultra(cid:173)
`violet for both sperm whale myoglobin and soybean
`leghaemoglobin a (see Fig. 2). As mentioned before,
`haem itself shows no fine structure bands between 250
`and 310 nm as evidenced by the difference spectrum
`for low-spin ferric haem cyanide versus high-spin
`ferric haem chloride. On the other hand, difference
`
`B
`
`0.12
`
`0 .10
`
`0.08
`
`0 .06
`
`0.04
`
`0 .02
`
`<,:
`<I
`
`0
`
`-002
`
`-0.04
`
`-0.06
`
`Wavelength (nm)
`Fig. l. Difference spectral titration of soybean ferric leghaemoglobin a with nicotinic acid ( A } in the near-ultraviolet and ( B) in the visible.
`Leghaemoglobin concentration was 33.4 µMin 0.01 M potassium phosphate buffer, pH 5.4, and each addition (1,2,3,4) increased the con(cid:173)
`centration of nictonic acid by 11 .6 µM . Difference spectra were performed with a four-cell arrangement that allowed blanking of both protein
`and ligand . Saturation was achieved at a final concentration of 58 µM nicotinic acid (not shown)
`
`Case No.: IPR2023-00206
`U.S. Patent No. 9,943,096
`
`Motif Exhibit 1135, Page 8 of 13
`
`

`

`136
`
`Structural Effects ofLiganding in Myoglobin and Leghaemoglobins
`
`8
`
`A
`
`I0 .05
`
`t>A
`
`hem in cyanide
`
`240
`
`340 360
`
`220
`
`240 260
`
`260 280 300 320
`3 20 3 40 360
`30 0
`280
`Wavelength (nm)
`Wavelength (nm )
`Fig . . 2. Form difference spectra for ( A ) sperm whale f erric my oglobin and ( BJ soybean ferric /eghaemoglobin a /iganded complexes. For (A)
`; for (B) LIA of 0.05 corresponds to ii Li e of 2.3 mM - 1 cm - 1 • All complexes were
`LIA of 0.05 corresponds to a Li e of 1.9 mM - 1 cm - 1
`measured against the corresponding acetate complex as reference. The difference spectra are labelled according to the bound ligand. Some
`of these difference spectra have been offset vertically for clarity and the baseline position for each marked with a dash through the curve
`on the long-wavelength side. The dashed curves between 270 and 295 nm are inferred background absorptions due to the haem. For reference,
`the curve (0- -0) for cyanoferric haem (haemin cyanide) versus haemin chloride is also shown. All samples were in 0.1 M sodium acetate
`buffer, pH 5.2, and liganded complexes were formed by titration with solid ligand until no further spectral changes were observed. Leghaemo(cid:173)
`globin a concentration was 22 µM and myoglobin concentration 26 µM at 20 °C. A four-cell arrangement was used
`
`spectra of both myoglobin and leghaemoglobin low(cid:173)
`spin ferric complexes versus the high-spin acetate
`complex do show fine structure difference bands in
`this spectral region and these must reflect changes in
`the environments of the aromatic amino acids upon
`such a transition . However, there is a difference be(cid:173)
`tween the responses ofmyoglobin and leghaemoglobin
`to a change in the sixth ligand.
`On formation of a low-spin complex from a high(cid:173)
`spin one in myoglobin there is a large difference ab(cid:173)
`sorption attributable to aromatic aminp acids (see
`especially the cyanide complex versus the acetate) with
`peaks at 280 and 286 nm. These are most likely to arise
`from the exposure of a buried tyrosine to aqueous
`solvent since tryptophan would give rise to additional
`peaks at higher wavelengths.
`Replacement of one high-spin ligand for another
`in myoglobin (fluoride or cyanate for acetate) shows
`a much smaller aromatic contribution to the difference
`spectrum. The azide complex of myoglobin which is
`mixed-spin but predominantly low-spin appears not.
`to follow the expected pattern if the proposed back(cid:173)
`ground absorption that we have drawn is correct.
`The form difference spectra for leghaemoglobin
`(Fig. 2B) show much smaller aromatic contributions
`than do those for myoglobin. The wavelengths of the
`difference peaks are, however, indicative of a change in
`the environment oftryptophan rather than of tyrosine,
`
`as was observed for the nicotinate versus aquoferric
`complex in Fig. 1 A. Since tryptophan difference
`spectra are expected to be three times larger than for
`tyrosine, given the same change in environment [13, 20],
`this means that the change in the tryptophan(s) en(cid:173)
`vironment in leghaemoglobins is minor compared to
`in myoglobins. Cyanate
`tyrosine(s)
`the
`that for
`(mixed-spin) and fluoride (high-spin) gave aromatic
`peaks nearly as intense as those given by cyanide (low(cid:173)
`spin), imidazole (low-spin) and azide (mixed-spin).
`
`Binding of Rose Bengal to the Apoproteins of
`Myoglobin and