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`R. Wegmann, Paris
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`H
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`

`

`CELLULAR AND MOLECULAR BIOLOG'
`
`Volume 46, Number 3
`
`THE ROLES OF METALS IN THE BRAIN, Part I
`
`NATIONAL LIBRARY OF MEDICINE
`
`Ill rn 111ll~1111111111
`
`NLM 01511928 8
`
`Content
`
`S.M.LEVINE
`
`M.B.H. YOUDIM ands. YEHUDA
`
`A. ESPINOSA DE LOS MONTEROS, R.A. KORSAK, T. TRAN,
`D: Vu, J. DE VELLIS and J. EDMOND
`
`J. HAN, J.R. DAY, K. THOMPSON, J.R. CONNOR
`and J.L. BEARD
`
`C.B. MARTA, 0.E. ESCOBAR CABRERA, C.l. GARCIA,
`M.J. VILLAR, J.M. PASQUINI and E.F. SOTO
`
`Z.M. QIAN, Q.K. LIAO, Y. To, Y. KE, Y.K. TsOI,
`G.F. WANG and K.P. Ho
`
`T. Moos, D. TRINDER and E.H. MORGAN
`
`K. WILLIAMS, M.A. WILSON and J. BRESSLER
`
`M.D.MAINES
`
`D. HAM and H.M. SCHIPPER
`
`K.R. WAGNER, Y. HUA, G.M. DE COURTEN-MYERS,
`J.P. BRODERICK, R.N. NISHIMURA, S.-Y. Lu
`and B.E. DWYER
`
`K. CHEN and M.D. MAINES
`
`M.R. EMERSON, F.E. SAMSON and T.L. PAZDERNIK
`
`M.V.R. KUMARI, M. HIRAMATSU and M. EBADI
`
`lll
`
`Forward
`
`491
`
`The neurochemical basis of cognitive deficits induced by
`brain iron deficiency: involvement of dopamine-opiate
`system. A review
`
`501
`
`517
`
`529
`
`541
`
`549
`
`563
`
`573
`
`587
`
`597
`
`609
`
`619
`
`627
`
`Dietary iron and the integrity of the developing rat brain: a ,
`study with the artificially-reared rat pup
`.
`
`Iron deficiency alters H- and L-ferritin expression in rat
`brain
`
`Oligodendroglial cell differentiation in, ra,_t .. brain is
`accelerated by the intracranial injection of apotransferrin
`
`Transferrin-bound and transferrin-free iron uptake by
`cultured rat astrocytes
`
`Cellular distribution of ferric iron, ferritin, transferrin and
`divalent metal transporter 1 (DMTl) in sub?tantia ni~
`and basal ganglia of normal and ~2-m1croglobulm
`deficient mouse brain
`
`Regulation and developmental expression of the divalent
`metal-ion transporter in the rat brain.
`
`The heme oxygenase system and its functions in the brain.
`A review
`
`Heme oxygenase-1 induction and mitoc:hondri.al iron
`sequestration in astroglia exposed to amylo1d peptides
`
`Tin-mesoporphyrin, a potent heme oxyge~ase. inhibito_r,
`for treatment of intracerebral hemorrhage: m vivo and m
`vitro studies
`
`Nitric oxide induces heme oxygenase-1 via mitogen(cid:173)
`activated protein kinases ERK and p38
`
`Effects of hypoxia preconditioning on expression of
`metallothionein-1,2 and heme oxygenase-1 before and
`after kainic acid-induced seizures
`
`Free radical scavenging actions of hippocampal
`metallothionein isofonns and of antimetallothioneins: an
`electron spin resonance spectroscopic study
`
`A.J. NAPPI and E. VASS
`
`637
`
`Iron, metalloenzymes and cytotoxic reactions. A review
`
`Indexed/ Abstracted in:
`Current Contents, Index Medicus,
`MEDLINE, BIOSJS Database, SUBIS,
`PASCALICNRS Database, Cam. Sci. Abstr.,
`CAB Inter., Chem. Abstr. Service, RIS in Reference
`
`Continued on the inside hackcover
`
`ISSN 0145-5680
`CMBIDI 46(3)491-698 (2000)
`
`Published by C.M.B. ASSOCIATION
`Editorial Office: 1, Avenue du Pav6 Neuf
`F·SJl 60 Noisy-le-Grand (France)
`
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`
`

`

`Cellular and
`Molecular BiologyTM
`
`Editor-in-Chief
`R.J. Wegmann
`Institut d'Histochimie Medicate des Universites de Paris, Paris
`
`Associate Editor, U.S.A. and Canada
`R.F. Ochillo
`Graduate School, Morgan State University, Baltimore, Maryland
`
`Associate Editor, Asia
`G. Yamada
`C.A.R.D., Kumamoto University, Kumamoto
`
`Associate Editor, Europe
`K.-J. Halbhuber
`Institut fiir Anatomie II, Universitii.t Schiller, Jena
`
`Guest Editor
`S.M. Le Vine
`University of Kansas Medical Center, Kansas City, Kansas
`
`

`

`Cellular and Molecular Biology™
`
`Disclaimer
`The Editor-in-Chief and the Editorial Board wish to make it clear that no inaccurate or misleading data,
`opinion and statement should appear in the articles and advertisements of this journal and that they are under
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`

`

`Cellular and Molecular Biologr 46 (3), 685-696
`Printed in France.
`
`0145-5680/00
`2000 Cell. mol. Biol.-
`
`:METAL-CATALYZED OXIDATION OF BRAIN-DERIVED
`NEUROTROPIIlC FACTOR (BDNF):
`SELECTIVITY AND CONFORMATIONAL CONSEQUENCES
`OF IDSTIDINE MODIFICATION
`
`Jana L. JENSEN1, Krzysztof KUCZERA 2, Suzanne ROY3 and Christian SCHONEICH16
`
`1th Department of Phannaceutical Chemistry, University of Kansas,
`2095 Constant Avenue, Lawrence, KS 66047, USA
`fax +I 785 864 5736; e-mail schoneic@ukans.edu
`2 Department of Chemistry and Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA
`3 Department of Product Development, Amgen Inc., Thousand Oaks, CA 91320, USA
`
`Received January 3, 2000; Accepted March 9, 2000
`
`Abstract - We have studied the metal-catalyzed oxidation (MCO) of brain-derived neurotrophic factor (BDNF) with
`regard to target sites and potential conformational changes of the protein. The exposure of BDNF to three different
`levels of ascorbate/Cu(II)/02 [20 µM Cu(m, 2 mM ascorbate (level I); 20 µM Cu(II), 4 mM ascorbate (level 2); 40
`µM Cu(II), 4 mM ascorbate (level 3)), chosen based on the extent of chemical modification of Met and His,
`respectively, resulted in the exclusive ox.idation of a buried Met residue, Met92, at level 1 but in the predominant
`oxidation of His at level 3. His modification had a significant impact on the structure of BDNF, as quantified by CD
`and ANSA fluorescence measurements, while Met ox.idation had not, also assessed through complementary oxidation
`of BDNF through hydrogen peroxide. Our ultimate objective was the correlation of the surface exposure of an
`oxidized His residue in a protein with potential effects on the conformational integrity of the oxidized protein. In a
`series of three proteins, human growth hormone (hGH), human relaxin (hRlx), and BDNF, we have now observed
`that His oxidation is paralleled by significant conformational changes when the target His residue is more surface
`exposed (hRlx, BDNF) while conformational consequences of His modification are less significant when the target
`His residues are more buried in the interior of the protein (hGH).
`
`Key words: Brain-derived neurotrophic factor (BDNF), metal-catalyzed oxidation (MCO), histidine and methionine
`oxidation, protein conformation
`
`Abbreviations: a.m.u.: atomic mass units; ANSA: 8-
`anilinonaphthalcne- I -sulfonic acid; BDNF: brain(cid:173)
`derived neurotrophic factor; CD: circular dichroism;
`EDTA: ethylenediaminetetraacetic acid; Endo Lys-C:
`electrospray
`endoproteinase Lys-C; ESI-MS:
`ionization mass spectrometry; HPLC/ESI-MS:
`reversed phase HPLC coupled on-line to ESI-MS;
`MALDI-TOF MS: matrix-assisted laser desorption
`ionization - time of flight mass spectrometry; MCO:
`metal-catalyzed oxidation; SASA: solvent accessible
`surface area; SDS: sodium dodecyl sulfate; SDS(cid:173)
`PAGE: sodium dodecyl sulfate polyacrylamide gel
`electrophoresis
`
`INTRODUCTION
`
`Redox active transition metals can catalyze the
`oxidation of proteins in the presence of electron
`donors (prooxidants), oxygen and/or peroxides
`(Levine, 1983a, 1983b; Farber and Levine, 1986;
`Rivett and Levine, 1990; Stadtman, 1990; Li et al.,
`1995; Schoneichetal., 1997; Zhao et al., 1997). Such
`metal-catalyzed oxidation (MCO) affects specifically
`amino acids involved in metal binding or located in
`
`685
`
`-685-
`
`
`
`
`

`

`686
`
`J.L. Jensen et al.
`
`close vicinity to metal binding sites. Often, the MCO
`of proteins leads to the loss of function and/or
`conformational changes. Biologically important
`prooxidants
`include
`ascorbate,
`thiols
`and
`hydroquinones (Miller et al., 1990). Their prooxidant
`activity results from the facile reduction of redox(cid:173)
`active transition metals. The redu~ed transition
`metals then react with molecular oxygen to generate
`a series of reactive oxygen species such as
`superoxide, hydrogen peroxide, and, potentially,
`hydroxyl radicals (or their metal-bound equivalents)
`(Miller et al., 1990; Yamazaki and Piette, 1991).
`
`Under normal physiological conditions, cellular
`levels of "free" redox-active transition metals such as
`Cu and Fe are very low (Lippard, 1999; Rae et al.,
`1999). However, certain pathological conditions are
`associated with elevated concentrations of redox(cid:173)
`active transition metals. For example, brain tissue of
`Alzheimer's disease patients was shown to contain
`higher levels of redox-active Fe (Smith et al., 1997).
`Increasing evidence is also mounting that the
`progression of amyotrophic lateral sclerosis (ALS) is
`caused by conformationally unstable SOD 1 mutants
`containing a Cu center which not only reacts with
`superoxide but also generates superoxide through the
`reaction with prooxidants such as ascorbate (Estevez
`et al., 1999). Moreover, these SOD 1 mutants may
`ultimately lose Cu to the environment. Hence,
`pathological conditions may promote the formation
`of a pool of redox-active transition metals available
`for binding to and reaction with biomolecules.
`
`The extent to which MCO can affect proteins will
`depend on the affinity for specific transition metals
`and the impact of oxidation on the integrity of the
`proteins. In our laboratory, we specifically focus on
`structure-reactivity relationships of protein oxidation
`and the question of how the oxidation of individual
`amino acids affects protein conformation. Recently,
`we observed that the Cu-catalyzed oxidation of His in
`two different proteins, human relaxin (hRlx) and
`human growth hormone (hGH), had a significantly
`different impact on these proteins. While hRlx
`experienced extensive structural changes, ultimately
`leading to non-covalent aggregation and a pH-
`
`dependent prec1p1tation (Li et al., 1995), such
`characteristics were not observed for hGH (Zhao et
`al., 1997). A possible rationale for this contrasting
`behavior is the position of the oxidation-sensitive His
`residue with regard to the tertiary structure of the
`protein, buried in hGH but more solvent exposed in
`Rlx. These observations suggest that the level of
`solvent exposure may be one parameter controlling
`the propensity for His oxidation to cause structural
`changes in the target protein. In order to fully
`rationalize the biological consequences of protein
`oxidation in vivo it is important that we understand
`not only the underlying mechanisms but also the
`parameters which determine the selectivity and
`conformational impacts of oxidative modifications.
`
`In the present paper, we have extended our studies to
`the Cu-catalyzed oxidation of an additional model
`protein, brain-derived neurotrophic factor (BDNF),
`in an effort to further elucidate the relationship
`between the location of target His residues and His
`oxidation on the integrity of higher-order protein
`structure. BDNF belongs
`to
`the
`family of
`neurotrophins which are important for neuronal
`survival and extracellular control of development and
`maintainance of neurons (Hofer and Barde, 1988;
`Robinson et al., 1995; Schuman, 1997; Estevez et al.,
`1999). Although BDNF does not contain a specific
`metal-binding site, it contains two highly surface(cid:173)
`exposed His residues, located at positions 1 and 75,
`displayed in fig. 1 (Leibrock et al., 1989; Rosenthal et
`al., 1991; Acklin et al., 1993). In order to separate the
`effects of His oxidation from Met oxidation, we
`compared BDNF exposed to MCO and hydrogen
`peroxide at three different levels, selected such that the
`overall yield of oxidized BDNF by both systems was
`comparable. The
`following concentrations of
`ascorbate and Cu(m were adjusted for MCO: 20 µM
`Cu(II), 2 mM ascorbate (level 1); 20 µM Cu(m, 4 mM
`ascorbate (level 2); 40 µM Cu(II), 4 mM ascorbate
`(level 3). In all cases, these levels of reactants are
`significantly higher compared to what would be found
`in vivo (typical physiological levels of ascorbate are on
`the order of a few µM; Rose and Bode, 1993).
`However, these higher concentrations were selected in
`order to perform the experiments in a reasonable time.
`
`\
`
`-686-
`
`
`
`
`

`

`Metal-catalyzed oxidation of BDNF
`
`687
`
`MATERIALS AND METHODS
`
`Materials
`Recombinant BDNF was provided by Amgen (Thousand Oaks,
`CA). The protein was exchanged into doubly distilled deionized
`water (dd. H10) via ultrafiltration using Microcon-3
`microconcentrators (3 k.Da cutoff) from Amicon (Beverly, MA).
`Typically, 500 µI of a 1 mg/ml BDNF solution in phosphate
`loaded onto a
`buffered saline (PBS), pH 7.4, was
`microconcentrator and centrifuged at 12,000 g for 50 min.,
`followed by three washes with 300 µI dd. H10. The retentate
`was collected and the BDNF concentration determined by UV
`spectroscopy (Shimadzu UV-160 spectrophotometer; Kyoto,
`Japan) using an extinction coefficient of 1.76 cm2/mg at 280 nm.
`The BDNF solution was aliquotted in amounts of 270 µg/vial
`which were dried in a vacuum centrifuge (Labconco Centrivap
`Concentrator, Kansas City, MO) and stored at -70°C for
`subsequent experiments. Sequencing grade endoproteinase Lys-C
`(Endo Lys-C) was obtained from Promega (Madison, WI). 8-
`Anilinonaphthalene-1-sulfonic acid (ANSA) was obtained from
`Sigma (St. Louis, MO). For amino acid analysis, 4N
`
`methanesulfonic acid, phenyl isothiocyanate and Amino Acid
`Standard H were from Pierce (Rockford, 11), ACS grade NaOH
`(filtered prior to use) was from Fisher (Pittsburgh, PA) and
`formic acid (99%) was from Fluka (Ronkonkoma, NY).
`
`Calculations of solvent exposure
`Solvent exposure was determined by calculating the solvent
`accessible surface area (SASA) for each His side chain. The
`SASA was defined by the center of a spherical probe of a given
`radius as it rolled over the surface defined by atomic van der
`Waals radii of the molecule. We used the program NACCESS
`2.1.1 (Department of Chemistry and Molecular Biology,
`University College, London, UK) to calculate atomic accessible
`surface areas corresponding to a probe of radius 1.4 A using the
`Lee and Richards' algorithm (Lee and Richards, 1971).
`Calculations were performed on an SGI INDIG02 workstation
`(SGI, Mountain View, CA). Molecular structures were taken
`directly from Protein Data Bank files: lHGU for human growth
`hormone, 6RLX for relaxin and lBND for BDNF. The relative
`SASAs for His were calculated based on an extended Ala-His(cid:173)
`Ala peptide as the reference point for 100% solvent exposure.
`
`Fig.1
`
`Representation of the primary structure of the E. cbli-derived BDNF monomer
`
`-687-
`
`
`
`
`

`

`688
`
`J.L. Jensen et al.
`
`Reaction conditions
`Oxidation reactions were conducted in aqueous phosphate
`buffer, pH 7.4, containing 20 µM BDNF which, at this
`concentration exists entirely as dimer (Radziejewski et al.,
`1992). The oxidation of 20 µM BDNF by Cu(Il)/ascorbate/02
`and hydrogen peroxide (H20 2) was performed at three different
`levels of reactant concentrations, as listed in table 1. These
`reaction conditions were chosen to obtain different extents of
`conversion of His and Met by MCO, and comparable extents of
`Met oxidation alone by H202. For MCO, 1-ml solutions were
`incubated for one hr. at 25°C. The reagents were added in the
`following order: buffer, BDNF, CuCli and ascorbate. After
`addition of CuCl2, the mixture was incubated for five min. at
`25°C to promote metal binding to the protein before the reaction
`was started by the addition of ascorbate. For control samples,
`dd. H20 was added in place of ascorbate. In some cases, the
`oxidation was stopped by the addition of a final concentration
`of 40 µM EDTA. For the oxidation of BDNF by H20i, 1-ml
`solutions were incubated for 3 hrs. at 25°C. Control samples did
`not contain H20 2. In this system, the oxidation was stopped by
`the addition of catalase to a final content of 10 units/ml. All
`solutions were made with dd. H20. Stock solutions of
`phosphate were treated with 5 g/l 00 ml Chelex (BIORAD,
`Hercules, CA) to minimize metal contamination. Stock
`solutions of CuC)i, ascorbate and H202 were freshly prepared
`prior to the reactions.
`
`Electrospray ionization mass spectrometry (ESI-MS)
`Samples were prepared for ESI-MS experiments by acetone(cid:173)
`HCI precipitation (Ozols, 1990), followed by dissolution in dd.
`H20 prior to analysis. ESI-MS experiments were performed on
`an Autospec-Q tandem hybrid mass spectrometer (VG
`Analytical, Manchester, UK) equipped with a Mark ill ESI
`source and an OPUS data system. Samples were trapped and
`desalted prior to ESI by washing the sample onto a trapping
`column ( 15 mm x 1 mm of polymeric beads with 400 A pores,
`Michrom BioResources, Auburn, CA) with 0.1 % acetic acid at
`250 µ1/min. and eluting retained sample into the ESI source
`with 70/30 methanol/0.1 % acetic acid at 10 µI/min. The relative
`amount of each BDNF species present after oxidation was
`determined by measuring the peak heights, using similar
`baselines for each spectrum.
`
`Table 1
`Reactant concentrations for different
`levels of oxidation
`
`MCO
`
`Oxidation
`Level
`
`CuCl2
`(µM)
`
`Ascorbate
`(mM)
`
`2
`3
`
`20
`20
`40
`
`2
`4
`4
`
`H202
`
`Hi02
`(mM)
`
`2
`3
`5
`
`\
`
`Proteolytic digestion
`Samples were prepared for digestion by ultrafiltration (three dd.
`H20 rinses at 12,000 g for 50 min. using Microcon-3
`rnicroconcentrators) and vacuum centrifugation. Approximately
`270 µg BDNF was dissolved in 0.2 M Tris buffer, pH 8.5,
`containing 6 M guanidine-HCl and 1 mM EDTA. 20 µl of 0.1
`M dithiothreitol was added and the sample was incubated for
`one hour at 45°C. After cooling to room temperature, 40 µl of
`0.1 M iodoacetic acid was added and the sample was placed in
`the dark for 30 min. to allow for carboxymethylation of the
`cysteine residues. The samples were then desalted by extensive
`ultrafiltration using Microcon-3 microconcentrators and dried
`by vacuum centrifugation. The reduced and alkylated BDNF
`was reconstituted in 0.15 M ammonium bicarbonate buffer
`containing 2.2 M urea. 1.5% (w/w) Endo Lys-C was added and
`the mixture was incubated for 12 hrs. at 37°C. At this point, an
`additional 0.75% (w/w) Endo Lys-C was added and the mixture
`incubated for 4 additional hrs. at 37°C. The theoretically
`expected Endo Lys-C fragments are given in table 2.
`
`Peptide mapping by RP-HP LC
`Analysis of peptide fragments from the proteolytic digestion of
`BDNF was performed on a Vydac Cl 8 column (4.6 x 250 mm)
`(Vydac, Hesperia, CA) at 60°C and 0.7 ml/min., using UV
`detection at 215 nm. A gradient from 0% to 50% acetonitrile
`with 0.1 % trifluoroacetic acid over 70 min. was employed for
`the separation and elution of the peptide fragments. Satisfactory
`signal-to-noise ratios for the separated fragments were achieved
`for injections of ~.5 nmol protein.
`
`MAWI-TOFMS
`Matrix-assisted laser desorption ionization-time of flight
`(MALDI-TOF) mass spectra were obtained on a Hewlett
`Packard model G2025A (Hewlett Packard, Palo Alto, CA),
`which typically measures molecular masses below 3 kDa with
`an accuracy of at least 0.1 %. Peaks from the peptide map were
`collected and vacuum centrifuged to dryness. Samples were
`reconstituted in acetonitrile/isopropanol/0.1 % trifluoroacetic
`
`Table 2
`BDNF
`
`Theoretical endo Lys-C fragments of
`
`Fragment
`
`Sequence
`
`Kl (-1-25)
`K2 (26-26)
`K3 (27-41)
`K4 (42-46)
`K5 (47-50)
`K6 (51-57)
`K7 (58-65)
`K8 (66-73)
`K9 (74-95)
`KlO (96-96)
`Kll (97-116)
`K12 (117-119)
`
`MHSDPARRGELSVCDSISEWVTAADK
`K
`TAVDMSGGTVTVLEK
`VPVSK
`GQLK
`QYFYETK
`CNPMGYTK
`EGCRGIDK
`RHWNSQCRTTQSYVRALTMDSK
`K
`RIGWRFIRIDTSCVCTLTIK
`RGR
`
`-688-
`
`
`
`
`

`

`Metal-catalyzed oxidation of BDNF
`
`689
`
`acid [1: 3: 2 (v/v/v)]. The matrix used was a standard solution of
`sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid) in
`acetonitrile/H20 (1: l (v/v)).
`
`HPLCIESl-MS
`RP-HPLC coupled on-line to ESI mass spectrometry was used
`to analyze the peptide maps. The reversed-phase assay for
`peptide mapping was optimized on a l x 250 mm Vydac C18
`column (Vydac, Hesperia, CA), which was coupled to the
`Autospec-Q tandem hybrid mass spectrometer. The column
`effluent was monitored with UV detection at 215 nm, and split
`post UV detector to 8 µI/min. into the ESI source. Mass spectral
`data were collected and analyzed on the OPUS data system.
`
`Amino acid analysis
`HCl hydrolysis is the most widely used technique for amino
`acid analysis; however,
`this method has significant
`inadequacies. Specifically, HCI hydrolysis causes loss of Trp
`residues (Ozols, 1990) and can cause conversion of methionine
`sulfoxide [Met(O)] back to methionine (Met) (Ray and
`Koshland, 1962; Floyd et al., 1963; Keutmann and Potts, 1969).
`As both Trp and Met are oxidation sensitive, it was necessary to
`obtain an
`accurate quantitation of
`these
`residues.
`Methanesulfonic acid hydrolysis was employed, as this method
`readily determines Trp and Met/Met(O) residues (Simpson et
`al., 1976). Samples were prepared for amino acid analysis by
`pe1forming an acetone-RC! precipitation. Precip~tated sampl~
`were dissolved in formic acid prior to hydrolysis. Hydrolysis
`and neutralization were conducted as described (Simpson et al.,
`1976). Briefly, protein samples were hydrolyzed with 4 N
`methanesulfonic acid at 115°C for 22 hrs., then neutralized with
`4 N NaOH prior to derivatization. Hydrolyzed amino acids were
`derivatized with phenyl isothiocyanate and separated on a
`Waters Spherisorb S50DS2 column (4.6 x 250 mm) (Waters
`Chromatography, Milford, MA) at 45°C and 0.8 ml/min., using
`UV detection at 254 nm.
`
`Protein assay
`Soluble protein concentration was detennined using the
`Coomassie® plus protein assay reagent from Pierce (Rockford,
`II), which is a modification of the Bradford method (Bradfor?,
`1976). Sample analysis involved the addition of the Coomass1e
`dye to the protein solution, with the subsequent measurement of
`absorbance at 595 nm on a Shimadzu UV-160 spectro(cid:173)
`photometer (Shimadzu, Kyoto, Japan).
`
`SDS-PAGE
`Samples were electrophoresed on a PhastSystem (Pharmacia
`Biotech, Piscataway, NJ) using precast PhastGel Gradient 8-
`25% acrylamide gels at 250 volts for ca. 1 hr. Approximately 0.8
`µg protein was loaded into each lane. Gels were stained using
`the PhastGel Silver Kit (Pharmacia Biotech).
`
`Analytical ultracentrifugation
`Sedimentation equilibrium and velocity experiments were
`performed on a Beckman XI.A analytical ultracentrifuge
`(Beckman Coulter, Fullerton, CA), equipped with an eight cell
`rotor. Samples were dialyzed extensively against 10 mM
`sodium phosphate buffer, pH 7.4, at 4 °C prior to centrifugation.
`
`For the sedimentation equilibrium experiments, samples were
`diluted with 10 mM sodium phosphate buffer, pH 7.4 to
`concentrations of 5, 10 and 20 µM. Samples were centrifuged at
`three different speeds (15,000, 20,000 and 27,000 rpm) for 24
`hrs. at 20°C. Equilibrium was confirmed by monitoring the UV
`absorbance at 229 nm, measured every 5 hrs. Raw data were
`imported into the software program Kdalton (developed by John
`Philo, Amgen) for analysis via non-linear curve fitting. For
`sedimentation velocity experiments, samples of 20 µM BDNF
`were equilibrated to 25°C and centrifuged at 40,000 rpm.
`Sedimentation was monitored using UV absorbance at 229 nm.
`The data generated were fit to a single species model using the
`modified Fujita-MacCosham function in the software program
`SVEDBERG, version 6.10 (developed by John Philo, Amgen).
`
`8-Anilino-l -naphtha.lenesulfonic acid ( ANSA) fluorescence
`Fluorescence spectra were ohtained using a Photon Technology
`International (Monmouth Junction, NJ) spectrofluorometer
`(Quanta Master Luminescence Spectrometer, QMl). ANSA
`(100 µM) containing 20 µM EDTA, was incubated with 10 µM
`BDNF (from either an oxidized or control sample) in 20 mM
`sodium phosphate buffer, pH 7.4, at 37°C for 30 min. The
`fluorescence emission spectra (excitation, 370 nm) were
`monitored between 400 and 600 nm. Binding of ANSA to
`protein was determined by subtracting the emission spectrum of
`ANSA from that of ANSA in the presence of BDNF.
`
`CD spectroscopy
`Samples were prepared for CD experiments by ultrafiltration
`and solvent exchange into dd. H20 (three dd. H20 rinses at
`12,000 g for 50 min. using Microcon-3 microconcentrators).
`Far-UV CD spectra (185-260 nm) were obtained on a Jasco J-
`720 spectropolarimeter (Jasco, Easton, MD), using a 0.1 cm
`path length quartz cell. The spectra were recorded at 25°C at a
`concentration of 100 µg/ml. Measurements were taken at
`intervals of 0.5 nm with a speed of 20 nm/min. and were
`averaged over 3 accumulated scans.
`
`RESULTS
`
`Solvent exposure of His
`The relative SASAs for the His residues of hGH are
`as follows: His18 - 37%, His21 - 26% and His151
`-
`89%. His18 and His21 were modified at levels of 63.5
`and 49.l %, respectively, by MCO (10 µM Cu(Il),
`100 µM ascorbate) while His151 was not (Zhao et al.,
`1997). The His residue in hRlx (position 12) has an
`SASA of 54%, and was modified 40% by MCO (200
`µM Cu(Il), 4 mM ascorbate) (Li et al., 1995). BDNF
`contains two His residues, one at the N-terminus and
`one at position 75. No X-ray coordinates are
`available for His1 (Robinson et al., 1995) indicating
`high flexibility and most likely high surface exposue.
`
`-689-
`
`
`
`
`

`

`690
`
`J.L. Jensen et al.
`
`His75 is characterized by an SASAof73%.
`
`Mass spectrometry
`ESI-MS experiments were performed to determine
`the extent of covalent modification of BDNF
`exposed
`to
`three different
`levels of
`the
`Cu(II)/ascorbate/02 and H20 2 systems, respectively.
`Fig. 2 shows representative mass spectra indicating
`the respective extents of BDNF oxidation at each
`oxidation level. The reactant concentrations and
`reaction times were selected such that they led to
`approximately similar extents of modification at each
`oxidation level, quantified in terms of oxygen
`
`incorporation into the protein. As revealed in fig. 2e,
`BDNF exposed to the harshest metal-catalyzed
`conditions (oxidation level 3) was extensively
`modified. Very little native BDNF (M.W. = 13637)
`remained, while significant amounts of species with
`molecular weights of 13653, 13669 and 13685 (all± 1)
`appeared. These molecular weights correspond to
`M+16, M+32 and M+48, respectively, indicating the
`incorporation of 1-3 equivalents of oxygen into the
`BDNF monomer (M =molecular weight of native
`BDNF). The oxidized species were not present in a
`control sample (data not shown). Table 3 summarizes
`the relative amounts ofBDNF species present at each
`
`13636
`(a)
`
`13636
`
`(b)
`
`13637
`
`(c)
`
`13653
`/13670
`
`(e)
`
`13653
`-13669
`-13685
`
`13637\
`
`13652
`
`13636
`
`13652
`
`(d)
`
`13669
`I
`
`13636
`
`(t)
`
`13668
`
`/13683
`
`ESl-MS spectra