After 1 h of incubation at 30C, 1 mL of 15% trichloroacetic acid was added to the reaction mixture of the total volume 5 mL; b Reaction with ninhydrin [24]: an aliquot 1 mL of GPAO/ DAPY
Trang 11,5-Diamino-2-pentyne is both a substrate and inactivator of plant copper amine oxidases
Zbyneˇk Lamplot1, Marek Sˇebela1, Michal Malonˇ2, Rene´ Lenobel2, Karel Lemr3, Jan Havlisˇ4, Pavel Pecˇ1, Chunhua Qiao5and Lawrence M Sayre5
1
Department of Biochemistry,2Laboratory of Growth Regulators, and3Department of Analytical Chemistry, Faculty of Science, Palacky´ University, Olomouc, Czech Republic;4Department of Analytical Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic;5Department of Chemistry, Case Western Reserve University, Cleveland, OH, USA
1,5-Diamino-2-pentyne (DAPY) was found to be a weak
substrate of grass pea (Lathyrus sativus, GPAO) and sainfoin
(Onobrychis viciifolia, OVAO) amine oxidases Prolonged
incubations, however, resulted in irreversible inhibition of
both enzymes For GPAO and OVAO, rates of inactivation
of 0.1–0.3 min)1were determined, the apparent KIvalues
(half-maximal inactivation) were of the order of 10)5M
DAPY was found to be a mechanism-based inhibitor of the
enzymes because the substrate cadaverine significantly
pre-vented irreversible inhibition The N1-methyl and N5-methyl
analogs of DAPY were tested with GPAO and were weaker
inactivators (especially the N5-methyl) than DAPY
Pro-longed incubations of GPAO or OVAO with DAPY
resul-ted in the appearance of a yellow–brown chromophore
(kmax¼ 310–325 nm depending on the working buffer)
Excitation at 310 nm was associated with emitted
fluores-cence with a maximum at 445 nm, suggestive of extended
conjugation After dialysis, the color intensity was
substan-tially decreased, indicating the formation of a low molecular
mass secondary product of turnover The compound pro-vided positive reactions with ninhydrin, 2-aminobenzalde-hyde and Kovacs’ reagents, suggesting the presence of an amino group and a nitrogen-containing heterocyclic struc-ture The secondary product was separated chromato-graphically and was found not to irreversibly inhibit GPAO
MS indicated an exact molecular mass (177.14 Da) and molecular formula (C10H15N3) Electrospray ionization- and MALDI-MS/MS analyses yielded fragment mass patterns consistent with the structure of a dihydropyridine derivative
of DAPY Finally, N-(2,3-dihydropyridinyl)-1,5-diamino-2-pentyne was identified by means of 1H- and 13C-NMR experiments This structure suggests a lysine modification chemistry that could be responsible for the observed inacti-vation
Keywords: amine oxidase; diamine; mechanism-based inhi-bition; nuclear magnetic resonance; oxidation
Copper-containing amine oxidases (CAOs, EC 1.4.3.6) play
a crucial role in the metabolism of primary amines These
enzymes are widely distributed in nature [1] In
micro-organisms, CAOs have a nutritional role in the utilization of primary amines as the sole nitrogen and carbon source In mammals and plants, CAOs appear to be tissue specific, and are implicated in wound healing, detoxification, cell growth, signaling and apoptosis [1] The oxidative deamination of amine substrates catalyzed by CAOs yields the correspond-ing aldehydes with the concomitant production of hydrogen peroxide and ammonia [2]
The reaction proceeds through a transamination mech-anism mediated by an active site cofactor topaquinone The cofactor is derived from the post-translational self-process-ing of a specific tyrosine residue that requires both active site copper and molecular oxygen [2] The key step in the oxidative deamination is conversion of the initial substrate Schiff base (quinoimine) to a product Schiff base (quino-aldimine) facilitated by Ca proton abstraction via a conserved aspartate residue acting as a general base at the active site [3] This step is followed by hydrolytic release of the aldehyde product and the reduced cofactor is finally reoxidized by molecular oxygen with the release of H2O2 and NH4+ The reduced topaquinone exists in two forms The first is an aminoresorcinol derivative coexisting with Cu(II), which is in equilibrium with the second form, Cu(I)-semiquinolamine radical [3] The role of copper in the reoxidation step has not been sufficiently elucidated for
Correspondence to M Sˇebela, Department of Biochemistry, Faculty of
Science, Palacky´ University, Sˇlechtitelu˚ 11, CZ-783 71 Olomouc,
Czech Republic Fax: + 420 5856 34933; Tel.: + 420 5856 34927;
E-mail: sebela@prfholnt.upol.cz
Abbreviations: ABA, 2-aminobenzaldehyde; ACA, 6-aminocaproic
acid; BEA, 2-bromoethylamine; CAO, copper-containing amine
oxidase; DABY, 1,4-diamino-2-butyne; DAPY,
1,5-diamino-2-pen-tyne; DDD, 3,5-diacetyl-2,6-dimethyl-1,4-dihydropyridine; DMAB,
4-(dimethylamino)benzaldehyde; DMAC,
4-(dimethylamino)cinna-maldehyde; ESI, electrospray ionization; GPAO, grass pea (Lathyrus
sativus) amine oxidase; HABA, 2-(4-hydroxyphenylazo)benzoic acid;
IT, ion trap; LSAO, lentil (Lens esculenta) amine oxidase; MALDI,
matrix-assisted laser desorption/ionization; OVAO, sainfoin
(Onobrychis viciifolia) amine oxidase; PSAO, pea (Pisum sativum)
seedling amine oxidase; PSD, post source decay; Q, quadrupole;
TNBS, 2,4,6-trinitrobenzenesulfonic acid.
Enzyme: copper-containing amine oxidase (EC 1.4.3.6).
Note: A website is available at http://prfholnt.upol.cz/biochhp
(Received 24 August 2004, revised 23 September 2004,
accepted 13 October 2004)
Trang 2most of the CAOs For the amine oxidase from Hansenula
polymorpha, it seems likely that cofactor reoxidation
involves electron transfer from substrate-reduced
topa-quinone to oxygen that is bound at a site separate from
copper [2]
Plant CAOs prefer diamine substrates like putrescine and
cadaverine, hence they are also called diamine oxidases [4]
Inhibitors of these enzymes have recently been reviewed [5]
Among them, a special place is reserved for
mechanism-based inhibitors, which undergo turnover-dependent
con-version to electrophilic products capable of covalent binding
to an active-site nucleophile resulting in inactivation Two
reported strategies in designing such inhibitors are the
incorporation of either halogen or unsaturation at the
b-position of amine substrates, examples being
2-bromo-ethylamine (BEA) [6] and 1,4-diamino-2-butyne (DABY)
[7], respectively Namely, the inactivating effect of DABY
(as a putrescine analog) on plant CAOs has been studied in
detail at the molecular level [8–10] Various b-unsaturated
compounds were tested in the reaction with bovine plasma
CAO [10–13] Propargylic and chloroallylic diamines were
highly potent inhibitors of the enzyme, more so than simple
allylic diamines [11–13] A recent study showed that the
homopropargyl amine, 1-amino-3-butyne, is also a potent
inactivator of certain CAOs [14] For this reason, and
because it is an analog of cadaverine (pentane-1,5-diamine),
the best known substrate of plant CAOs [4], it seemed
important to determine the potential inactivating properties
of the higher DABY homolog, 1,5-diamino-2-pentyne
(DAPY) The unsymmetrical DAPY comprises both a
propargyl and homopropargyl amine
DAPY was synthesized and tested as a substrate of two
plant CAOs DAPY acts as a mechanism-based inhibitor of
the enzymes, causing their modification with the
concom-itant inactivation However, in comparison with the effect of
DABY previously published [8], the modification extent is
considerably decreased Only a few amino acid side chains
seem to be modified as a result of the reaction A major part
of DAPY oxidation product, aminopentynal, after the
conjugate addition of an unreacted DAPY molecule, is
converted to a free nitrogenous heterocyclic compound,
whose dihydropyridine-derived structure was determined
using various analytical methods
Materials and methods
Chemicals
The previously unreported DAPY dihydrochloride was
synthesized from the known 1,5-dichloro-2-pentyne [15] by
displacement of the activated propargylic chloride with
methanolic ammonia in a pressure bottle [11],
tert-butoxy-carbonyl protection of the introduced primary amine group,
displacement of the less reactive homopropargylic chloride
with methanolic ammonia in a pressure bottle
(accompan-ied by an elimination side reaction), and finally
HCl-mediated deprotection Elemental analysis showed 33.38%
C, 7.41% H and 14.01% N (calculated 33.01, 7.08,
and 13.18%, respectively) Melting point: 177–179C;
13C-NMR spectrum (deuterium oxide): d 17.0, 29.4, 38.0,
74.3 and 82.8 p.p.m.; electrospray ionization ion trap mass
spectrometry (ESI-IT-MS) and MS/MS: a single
quasimo-lecular peak of m/z 99.1 providing fragment peaks of m/z 82.0 and 70.0 N5-Methyl-1,5-diamino-2-pentyne dihydro-chloride was prepared as for DAPY, substituting metha-nolic methylamine in the penultimate step N1 -Methyl-1,5-diamino-2-pentyne dihydrochloride was prepared by reac-tion of 1,5-dichloro-2-pentyne with aqueous methylamine, and then with methanolic ammonia Synthetic details and characterization of these analogs are given elsewhere [16] 3,5-Diacetyl-2,6-dimethyl-1,4-dihydropyridine (DDD) was prepared using the Hantzsch synthesis [17] 2-Amino-benzaldehyde (ABA), bicinchoninic acid solution (Cat No B9643), 4-(dimethylamino)benzaldehyde (DMAB), 4-(di-methylamino)cinnamaldehyde (DMAC), 3-hydroxypyri-dine, pyrrole and 2,4,6-trinitrobenzenesulfonic acid (TNBS) solution (5%, w/v) were from Sigma (St Louis,
MO, USA) Deuterium oxide (D2O, 99.96%) and d4 -methanol (CD3OD, 99.95%) were from Aldrich (Milwau-kee, WI, USA) 6-Aminocaproic acid (ACA) and NADH were supplied by Fluka (Buchs, Switzerland) 2-(4-Hy-droxyphenylazo)benzoic acid (HABA) was from Bruker Daltonik GmbH (Bremen, Germany) All other chemicals were of analytical purity grade
Enzymes Plant diamine oxidases from grass pea (Lathyrus sativus, GPAO) and sainfoin (Onobrychis viciifolia, OVAO) seed-lings were prepared in homogeneous forms following published protocols [18,19] Specific activities assayed with cadaverine as a substrate were 50 and 120 UÆmg)1, respect-ively Bovine liver catalase (2000 UÆmg)1) and horseradish peroxidase (100 UÆmg)1) were commercial products from Fluka Protein content in enzyme samples was estimated using a standard method with bicinchoninic acid [20]
Kinetic measurements CAO assay was carried out following a previously published protocol [9] The guaiacol spectrophotometric method was used, which is based on a coupled reaction of horseradish peroxidase [21] Kinetic parameters of time-dependent inactivation of the enzymes by DAPY were evaluated according to the literature on mechanism-based inhibition [11,22] Various 0.1Mpotassium phosphate buffers in the pH range 5.0–8.0 were used in experiments performed to describe the influence of pH on the inhibition potency of DAPY To assess the influence of ionic strength on the reaction, 0.1M Britton–Robinson buffer, pH 7.2, containing variable potassium chloride was used Rapid scanning of absorption spectra of GPAO or OVAO mixed with DAPY under admission of air was carried out by means of a DU-4500 spectrophotometer (Beckman, Fullerton, CA, USA) essen-tially as described previously [9] Aerobic scans at longer time intervals (10–120 min) after mixing GPAO or OVAO with DAPY (1 : 100) were carried out using a Lambda 11 spectrophotometer (Perkin–Elmer, Ueberlingen, Germany)
TLC of DAPY oxidation product TLC of the GPAO-DAPY reaction mixture was carried out using commercial TLC plastic sheets (4· 8 cm) with a layer of Silica gel 60 F (Merck, Darmstadt, Germany);
Trang 3n-propanol/MeOH/saturated sodium acetate solution
(40 : 3 : 60 v/v/v) was used as a mobile phase Primary,
secondary and tertiary amino groups were detected using
ninhydrin, sodium nitroprusside and Dragendorff’s
rea-gents, respectively Aldehyde groups were detected using
Schiff’s reagent
Spectrofluorimetry
A solution of DAPY (5 mM) in 20 mM potassium
phos-phate buffer, pH 7.0, was oxidized by an excess of GPAO at
23C for 12 h After that, the reaction mixture was filtered
using a centrifugal cartridge Microcon (Millipore, Bedford,
MA, USA), 0.5 mL, equipped with a 10 kDa cut-off filter
The filtrate was used for spectrofluorimetry Fluorescence
emission spectra of the DAPY oxidation product and
model compounds (DDD, 3-hydroxypyridine, NADH and
pyrrole) were obtained by means of an LS50B
spectroflu-orimeter (Perkin–Elmer, Boston, MA, USA) The oxidized
DAPY was measured with a fixed excitation at 310 nm
Similarly, for the model compounds, the respective
wave-lengths of maximal absorption were taken as excitation
wavelengths
Colorimetric trapping of DAPY oxidation product
For the various methods listed, absorption spectra were
recorded on Lambda 11 spectrophotometer against a blank
without DAPY (a) Reaction with ABA [23]: DAPY
(2.5 mM) was oxidized by an excess of GPAO (500 nkat)
in 0.1Mpotassium phosphate buffer, pH 7.0, in the presence
of catalase (200 U) and 2.5 mM ABA After 1 h of
incubation at 30C, 1 mL of 15% trichloroacetic acid was
added to the reaction mixture of the total volume 5 mL; (b)
Reaction with ninhydrin [24]: an aliquot (1 mL) of GPAO/
DAPY mixture (5 lM GPAO, 4 mM DAPY; initial
con-centrations) in 0.1Mpotassium phosphate buffer, pH 7.0,
was taken out after 2 h of incubation at 23C and mixed
with the same volume of warm ninhydrin reagent [24] This
was followed by the addition of acetic acid (1.5 mL) The
sample was kept in a boiling water bath for 30 min to
develop the color It was then cooled and 2.5 mL of acetic
acid was added to make up the volume to 6 mL (c)
Reac-tion with Kovacs’ reagent: an aliquot (1 mL) of GPAO/
DAPY mixture (2 lM GPAO, 0.2 mM DAPY; initial
concentrations) in 0.1M potassium phosphate buffer,
pH 7.0, was removed after 90 min of incubation at 23C
and mixed with 2 mL of the original Kovacs’ reagent
containing DMAB [7,8,25] (or its alternative contaning
DMAC [8]), incubated at 50C for 30 min and cooled on ice
bath In an alternative experiment, the GPAO/DAPY
reaction mixture was first separated by ultrafiltration using
the Microcon centrifugal cartridge as described above and
only the ultrafiltrate mixed with Kovacs’ reagent Three
model compounds (DDD, pyrrole and NADH) were used to
compare spectral properties of their DMAB-adducts with
that of the DAPY oxidation product
MS of DAPY reaction mixture
Samples for ESI-IT-MS were prepared by the oxidation of
5 m buffered DAPY solution with an excess of GPAO
Two buffers were used to optimize results: 0.1M ammo-nium bicarbonate, pH 7.8, and 0.1MBistris/HCl, pH 7.0
To evaluate the reactivity of the initial product aminoalde-hyde, the reaction was also carried out in the presence of
5 mM ACA as a trapping nucleophilic reagent After incubation at 30C for a sufficiently long time interval (6–24 h), the reaction mixtures were separated by ultrafil-tration using the Microcon centrifugal cartridge as given above The filtrate was properly diluted using methanol before measurements Mass spectra were obtained using an ion trap mass spectrometer Finnigan MAT LCQ (Thermo Electron Corp., San Jose, CA, USA) equipped with an ESI interface All samples were directly introduced to the electrospray interface of the mass spectrometer by a syringe
at a flow rate of 5 lLÆmin)1 The ionization mode used produced positively charged quasimolecular ions [M+H]+ Parameters of the electrospray were as follows: source voltage 5.6 kV, sheath gas flow 20 units, cone voltage 33.43 V, capillary temperature 250C
Enzymatic microscale production of DAPY oxidation product
The larger quantity of DAPY oxidation product required for further characterization was prepared by a cyclic flux
of DAPY solution through a hydroxyapatite column (1· 10 cm) containing immobilized GPAO and catalase After GPAO (10 mkat) and catalase (10 mkat) were loaded
in 10 mMammonium bicarbonate, pH 7.8, the column was washed with the same buffer Then 50 mL of 5 mMDAPY
in 10 mM ammonium bicarbonate was left to circulate through the column at 21C using a peristaltic pump at a flow rate of 1 mLÆmin)1for 24 h After stopping the cyclic flux, the column was additionally washed with 20 mL of
10 mMammonium bicarbonate, and the eluate was added
to the solution of oxidized DAPY The combined solution was then filtered using an ultrafiltration cell (100 mL) equipped with a 10 kDa cut-off filter (Amicon, Danvers,
MA, USA) Water was removed on a rotary vacuum evaporator Rotavapor R-200 (Bu¨chi, Switzerland) at 70 C The remaining solid was extracted by methanol (2· 1 mL), the extract transferred to a test tube and the solvent spontaneously evaporated at 21C Alternatively, an excess
of GPAO was added to 10 mL of 20 mMDAPY solution
in 20 mM potassium phosphate buffer, pH 7.0, and the resulting mixture incubated at 37C for 24 h During that time, GPAO was added twice more at 4-h intervals After ultrafiltration, the sample was processed as above
RP-HPLC separation of DAPY oxidation product The isolated DAPY oxidation product was dissolved in 0.3% (v/v) triethylamine acetate, pH 7.0 It was then separated by RP-HPLC on a Supelcosil LC18 column,
25 cm· 4.6 mm i.d., 5 lm particles (Supelco, Bellefonte,
PA, USA) connected to a Gold Nouveau 125 NM HPLC system equipped with a diode array detector Model 168 operating at 200–600 nm (Beckman, Fullerton, CA, USA) The buffers used were as follows: A, 0.3% (v/v) triethyl-amine acetate, pH 7.0; B, 0.3% (v/v) triethyltriethyl-amine acetate,
pH 7.0, containing 60% (v/v) acetonitrile Separations at
a flow rate of 1 mLÆmin)1 were run isocratically in the
Trang 4beginning (10 min), then with an increasing linear gradient
from 0 to 100% B for 20 min and isocratically at 100% B
for an additional 20 min This was followed with a
decreasing linear gradient from 100 to 0% B in 8 min and
a short final isocratic step to give the total time 60 min
Fractions showing highest absorption at 310 nm were
pooled, frozen and lyophilized For MALDI-MS and
ESI-MS analyses, the obtained solids were extracted by
methanol; for 1H-NMR experiments the extraction was
performed using D2O
MS of HPLC-separated DAPY oxidation product
MALDI-TOF-MS and MALDI-PSD-TOF-MS (PSD,
post source decay) were carried out using an Axima CFR
mass spectrometer (Kratos Analytical, Manchester, UK)
equipped with a nitrogen laser wavelength of 337 nm Peak
power was 6.0 mW: positive mode with pulsed extraction
was used MALDI probes were prepared by mixing 0.5 lL
of a sample diluted by acetonitrile with 0.5 lL of saturated
HABA in the same solvent Acquired spectra were
processed by Kratos Axima CFR software KOMPACT
v 2.1.1 Exact mass measurements to determine the
elemental composition of the DAPY oxidation product
were performed using ESI-Q-TOF-MS on a Q-Tof
microTM mass spectrometer (Micromass, Manchester,
UK) The collision-induced dissociation was used to get
MS/MS data All samples were directly introduced to the
electrospray interface of the instrument by a syringe at a flow
rate of 5 lLÆmin)1 The ionization mode used produced
positively charged quasimolecular ions [M+H]+
Parame-ters of the electrospray were as follows: source voltage
2.5 kV, cone voltage 15 V, source temperature 80C,
desolvation temperature 120C Acquired spectra were
processed byMASSLYNXv 4 software (Micromass)
NMR spectroscopy of DAPY oxidation product
First, GPAO-catalyzed oxidation of DAPY was carried
out in 20 mM D2O-potassium phosphate buffer, pD 7.0,
similarly to previous work performed with a CAO and
agmatine [26] DAPY.2HCl (3 mg) was dissolved in 0.5 mL
of D2O Control1H- and13C-NMR spectra were recorded
at 27C on a Bruker AVANCE 300 MHz NMR
spectro-meter (Bruker Analytik, Rheinstetten, Germany), using
tetramethylsilane as internal standard After this
measure-ment, the DAPY solution was pipetted into a test tube
containing a GPAO sample lyophilized from 0.5 mL of a
homogeneous GPAO (16 mgÆmL)1) in 20 mM potassium
phosphate buffer, pH 7.0 The mixture was shaken well on a
vortex and incubated at 30C for 12 h The enzyme protein
was then removed using the Microcon centrifugal cartridge
as described above, and the filtrate was used for recording
1H- and13C-NMR spectra.1H-NMR spectra in D2O were
also measured with the DAPY oxidation product extracted
from a lyophilizate obtained after RP-HPLC separation of
the GPAO/DAPY reaction mixture
Another NMR experiment was carried out as follows:
DAPY (5 mM) in 2 mL of 20 mM potassium phosphate
buffer, pH 7.0, was mixed with an excess of GPAO (5 mg,
added as a concentrated solution in the same buffer) and the
mixture was incubated at 30C for 12 h After that, the
same amount of GPAO was added again and the incubation proceeded for an additional 12 h Before1H- and13C-NMR measurements, the resulting solution was ultrafiltered as described above and the filtrate was lyophilized The NMR sample was prepared by extracting the lyophilizate with 0.5 mL of CD3OD
Determination of free primary amino groups Primary amino groups in GPAO were determined by modification of the established TNBS method [27,28] A sample of the enzyme (0.1 mL of a buffered solution containing 10–20 mgÆmL)1) was added to 0.9 mL of 4% (w/v) sodium bicarbonate, pH 8.5, in a test tube and mixed using a vortex Later, 0.5 mL of 0.01% TNBS was added with mixing and incubation at 40C in the dark for 1 h ACA (1 mgÆmL)1) was used as a standard to construct the corresponding calibration curve (10–50 lg) After incuba-tion, all samples were measured at 345 nm against a blank containing water instead of the enzyme To determine free amino groups in DAPY-reacted GPAO (100 : 1; 2 h of incubation at 30C), the reacted enzyme was exhaustively dialyzed against 20 mM potassium phosphate, pH 7.0, before an aliquot was processed as given above
Chromatofocusing, quinone staining Chromatofocusing was performed on a Mono P HR 5/20 column (Amersham Biosciences) connected to a BioLogic Duo Flow liquid chromatograph (Bio-Rad, Hercules, CA, USA) Loading buffer: 25 mM Tris/HCl, pH 8.2; elution buffer: Polybuffer 96 (Amersham Biosciences, 3 mL) was mixed with Polybuffer 74 (Amersham Biosciences, 7 mL), diluted with water, adjusted to pH 5.0 with acetic acid and then filled to a final volume of 100 mL All samples were dialyzed against the loading buffer before separation Redox-cycling quinone staining on nitrocellulose membrane was carried out as described previously [29]
Results
Kinetic measurements The oxidative conversion of DAPY was studied using two plant CAOs after these enzymes had been isolated from grass pea and sainfoin seedlings Initial rates measured with 2.5 mM DAPY showed that the compound is a weak substrate For GPAO, the initial rate reached 5% of the value measured for putrescine at the same concentration For OVAO, the initial rate with DAPY was 10% towards that of cadaverine as the best substrate for this enzyme However, because OVAO prefers cadaverine to putrescine
by a factor 2.7 (and such a property is unique among plant CAOs) [19], this value may be recalculated as 27% towards that of putrescine
Longer incubations of both studied enzymes with DAPY led to a significant decrease in their catalytic activity toward
normal substrates The inhibition was time- and concen-tration-dependent and irreversible, as the activity could not
be restored by dilution or dialysis Pseudo-first-order inhibition kinetics were observed at 30C with DAPY concentrations ranging from 5 to 40 l ; Fig 1 shows
Trang 5semilogarithmic plots for GPAO, where the slope for each
regression line represents the observed rate constant kobs
The kinetic constants describing the inactivation of GPAO
and OVAO were determined from the corresponding Kitz–
Wilson replots (1/kobsvs 1/[DAPY]; see inset in Fig 1 as an
example) From these plots kinact, the maximal rate of
inactivation, is 1/y) intercept, and KI, the concentration
required for half-maximal inactivation, is)1/x ) intercept
The determined values for GPAO were similar when
measured in 0.1M potassium phosphate or Bistris/HCl
buffers, pH 7.0 (Table 1) Comparatively, for OVAO,
inactivation by DAPY is slower, but the KIis lower
GPAO and OVAO (both 70 nM in 0.1M potassium
phosphate buffer, pH 7.0) were each individually incubated
with seven different concentrations of DAPY varying from
1 to 50 lM at 30C for 1 h Remaining activity was
determined by the ratio of the measured activity of the
inactivated enzyme to the control enzyme incubated without
DAPY A plot of the remaining activity (%) vs [DAPY]/
[GPAO] or [DAPY]/[OVAO] was constructed
Extrapola-tion of the linear porExtrapola-tion of the data at lower [DAPY] gave
the partition ratio (turnover number minus one) This ratio,
the number of molecules leading to product per each
inactivation event, was determined to be 120 for DAPY/ GPAO (Fig 2) and 200 for DAPY/OVAO
The inhibition strength of DAPY is dependent on pH GPAO (70 nM) was incubated with 50 lM DAPY in 0.1 potassium phosphate buffers of different pH values over the range 5.0–8.0 at 30C for 1 h The obtained remaining activity values were then plotted against pH DAPY showed
a maximal inhibition effect at pH 7.5 The extent of GPAO inhibition by DAPY is also influenced by ionic strength The reaction was performed in 0.1M Britton–Robinson buffer, pH 7.2, where ionic strength had been adjusted with KCl to reach values from the range 0.085–0.4 The percentage of remaining activity after 1 h of incubation of the reaction mixture (70 nM GPAO, 50 lM DAPY) at
30C increases with increasing ionic strength (not shown) Enzymes are protected against mechanism-based inhi-bitors by their substrates and competitive inhiinhi-bitors These compounds bind at the active site and compete with binding
of the inhibitor Inactivation of the enzyme is therefore slowed down GPAO (2 lM) was incubated with 100 lM DAPY in the absence and presence of 1 mMcadaverine as a substrate At chosen time intervals, aliquots of the reaction mixtures were taken out for activity assay The protective effect of cadaverine was significant For example, after
15 min of incubation, the remaining activity was 15% in the reaction mixture with cadaverine and only 8% without Due to the potential information that might be provided about the mechanism of enzyme inactivation by DAPY, we also determined the kinetics of inactivation of GPAO by the two possible N-monomethyl analogs of DAPY As shown
in Table 1, N1-methyl-DAPY and especially N5 -methyl-DAPY were weaker inactivators relative to -methyl-DAPY itself
Spectrophotometry and spectrofluorimetry, TLC Substrates of CAOs are known to disturb the characteristic absorption spectrum of the enzymes [4] Under anaero-biosis, the topaquinone cofactor maximum at 500 nm is bleached after the substrate addition and replaced by a complex spectrum of the Cu(I)-semiquinolamine radical showing maxima at 360, 435 and 465 nm This is supple-mented with a peak at 315 nm that is thought to reflect the
Fig 1 Effect of incubation time on inactivation of GPAO by DAPY.
The semilogarithmic plot was constructed for the following DAPY
concentrations: 5 (j), 10 (m), 20 (r) and 40 l M (d) Activity was
measured with 70 n M enzyme in 0.1 M potassium phosphate buffer,
pH 7.0, at 30 C by means of the guaiacol spectrophotometric method
[21] The inset shows the corresponding Kitz–Wilson replot for the
determination of k inact and K I values.
Table 1 Inactivation kinetics Experiments were performed at 30 C in
0.1 M potassium phosphate buffer, pH 7.0, except where noted.
Inhibitor/enzyme
k inact
(min)1)
K I
(l M )
t 1/2 at saturation a
(min) DAPY with GPAO b 0.27 45 2.2
DAPY with GPAO 0.31 50 1.9
DAPY with OVAO 0.13 10 4.5
N1-Methyl-DAPY
with GPAO
0.11 45 6.3
N5-Methyl-DAPY
with GPAO
0.05 36 13.9
a Time required for half of the enzyme to become inactivated in the
presence of saturating concentration of inhibitor.bIn 0.1 M Bistris/
HCl buffer, pH 7.0.
Fig 2 Partition ratio plot for inactivation of GPAO by DAPY Residual GPAO activities after 1 h of incubation with DAPY were plotted against the corresponding values of the concentration ratio [DAPY]/[GPAO] Activity was measured with 70 n M enzyme in 0.1 M
potassium phosphate buffer, pH 7.0, at 30 C using the guaiacol spectrophotometric method [21].
Trang 6presence of the aminoresorcinol form of topaquinone (the
fully reduced cofactor), which is in equilibrium Using
rapid-scanning techniques, the mentioned spectral features
are observable also in the presence of air [9]
As shown in Fig 3 (upper), rapid scanning after the
aerobic addition of DAPY to a purified GPAO in 0.1M
potassium phosphate buffer, pH 7.0, revealed the formation
of a spectrum identical to that of a substrate-reduced CAO
In addition, the reaction of the studied plant CAOs with
DAPY gave rise to an oxidation product providing near
UV/visual absorption with a maximum at 310–315 nm,
which increased in intensity with temperature Absorbances
measured after 90 min of incubation of GPAO/DAPY
reaction mixtures at 50C were almost three times higher
than those measured after the incubation at 37C Figure 3
(lower), shows an increasing development of the product
within the first 10 min after mixing OVAO with DAPY in
0.1M potassium phosphate buffer, pH 7.0 The same
spectrum was also observable in the GPAO/DAPY reaction
mixture In 0.1MBistris/HCl buffer, pH 7.0, the product
absorption maximum was shifted to 325 nm (not shown)
GPAO and OVAO were fully inactivated by incubation
with an excess of DAPY and no activity could be recovered
by dialysis The inactivated enzymes after dialysis were still
faint yellow due to a broad absorption below 330 nm, but
the color intensity was substantially decreased
After separation of the enzyme protein by ultrafiltration,
the GPAO/DAPY reaction mixture exhibited a fluorescence
emission spectrum with a maximum at 445 nm (shoulders at
485 and 520 nm) when excited at 310 nm For model compounds, the following fluorescence characteristics were obtained: DDD, solvent water, emission maximum at
503 nm (excitation at 410 nm); 3-hydroxypyridine, solvent water, emission maximum at 460 nm (excitation at
310 nm); NADH, solvent water, emission maximum at
465 nm (excitation at 340 nm); pyrrole, solvent water, emission maximum at 360 nm (excitation at 290 nm) TLC experiments revealed the presence of a free primary amino group in the DAPY oxidation product obtained by the reaction of GPAO (positive ninhydrin spot, Rf¼ 0.62); DAPY itself showed Rf¼ 0.33 in the same system Staining for tertiary amines (Draggendorff’s reagent) was also positive for the GPAO/DAPY reaction mixture (an orange spot, Rf¼ 0.62) Staining for aldehydes using Schiff’s reagent was negative
Colorimetric detections of DAPY oxidation product Kovacs’ reagent containing DMAB (detects indoles and pyrroles [25]) was previously used for the visualization of pyrrole derivatives formed by the oxidation of DABY by plant amine oxidases [7,8] Reaction mixtures of the studied CAOs with DAPY reacted positively with Kovacs’ reagent after a period of incubation and provided a red soluble adduct upon heating The red color intensity increased with increasing temperature in the range 30–50C A typical absorption spectrum of the adduct with a maximum at
520 nm (shoulder at 500 nm) is shown in Fig 4 (upper)
Fig 3 Spectrophotometric studies on the
reactions of GPAO and OVAO with DAPY.
(Upper) Difference absorption spectrum of
GPAO (20 l M ) after the addition of DAPY
(final concentration 1 m M ) in air-saturated
0.1 M potassium phosphate buffer, pH 7.0.
The spectrum was recorded 2 s after mixing
the reactants at 30 C, using the
rapid-scan-ning technique [9] (Lower) Time-dependent
development of the DAPY oxidation product
as observed in difference absorption spectra.
The spectra were recorded using a solution of
OVAO (20 l M ) in air-saturated 0.1 M
potas-sium phosphate buffer, pH 7.0, after adding
0.1 M DAPY (1 m M final concentration) at
30 C Intervals between scans: 15 s, total
time: 10 min.
Trang 7Replacing DMAB in Kovacs’ reagent with DMAC led to a
shift of the adduct absorption maximum to 650 nm (not
shown) However, if the reaction mixture was dialyzed
before the addition of the reagent, the spectrum was almost
negligible (Fig 4, upper) Three model compounds were
tested for this reaction The synthesized DDD reacted with
Kovacs’ reagent to form a product with an absorption
maximum at 600 nm having a shoulder at 560 nm (Fig 4,
lower) NADH also reacted with the reagent and provided a
spectrum with a single peak centered at 510 nm
DMAB-reacted pyrrole provided a maximum at 565 nm with a
shoulder at 520 nm
The use of ABA for the spectrophotometric activity assay
of plant CAOs was refined almost four decades ago [23]
Plant CAOs oxidize the diamines putrescine and cadaverine
to form the corresponding aminoaldehydes, which sponta-neously cyclize to 1-pyrroline and 1-piperideine, respect-ively The latter cyclic imines condense with ABA to generate the corresponding substituted dihydroquinazolin-ium compounds The DAPY oxidation product obtained
by the reaction of GPAO provided an adduct with ABA characterized by an absorption maximum at 430 nm (not shown)
The ninhydrin reagent described for activity assay of CAOs by Naik et al [24] was also tested to trap the DAPY oxidation product in the reaction mixture with GPAO The same cyclic imines above (1-pyrroline and 1-piperideine) react with ninhydrin in strongly acidic medium to form colored compounds of unknown structure with absorption maxima at 440 and 515 nm, respectively [30] The DAPY oxidation product displayed a broad absorption between
400 and 550 nm with a maximum at 465 nm after reaction with ninhydrin (not shown)
MS of DAPY reaction mixture Figure 5 (upper) shows an ESI-IT mass spectrum of the GPAO/DAPY reaction mixture prepared using 0.1M ammonium bicarbonate, pH 7.8 There are two major peaks of the reaction product observable in the spectrum with m/z 178.3 and 222.3 The former ion showed fragment peaks with m/z 161.3, 149.2 and 135.2, the latter provided peaks with m/z 205.2, 193.2 and 176.2 in the respective MS/MS spectra The peak with m/z 222.3 was not observed when the reaction was carried out in 0.1M Bistris/HCl buffer, pH 7.0 Figure 5 (lower) shows a mass spectrum of the GPAO/DAPY reaction mixture prepared in 0.1M ammonium bicarbonate containing ACA as a reagent for trapping of the product aminoaldehyde, where several new peaks appeared ACA itself is represented by a peak with m/z 132.1 (MS/MS: a clear fragment peak with m/z 114.1.) There is one more peak visible with m/z 211.3 (MS/MS: fragment peaks with m/z 193.3, 106.0 and 96.0), which probably reflects an adduct of the reaction product with ACA
ESI-IT-MS of the low molecular mass fraction of the GPAO/DAPY reaction mixture prepared in 20 mM potassium phosphate buffer, pD 7.0 (made in D2O for the purpose of NMR spectroscopic analysis) revealed isotopic peaks belonging to quasimolecular ions of the reaction product The highest intensity was observed for
a peak with m/z 179.2, lower intensities were observed for peaks in the following order: m/z 181.2, 180.2, 182.2 and 178.2 The peak with m/z 179.2 provided an MS/MS spectrum showing fragments with m/z 162.2, 150.2 and 136.2 (not shown)
HPLC separation and MS analysis of DAPY oxidation product
HPLC separation of the isolated DAPY oxidation product from enzymatic microscale production was carried out using an instrument equipped with a diode array detector Thus individual runs could be monitored continuously at
214, 240 and 310 nm The buffer system used was chosen according to that published for peptide separation from tryptic digests [31]
Fig 4 Reaction of the DAPY oxidation product and a dihydropyridine
model compound with 4-(dimethylamino)benzaldehyde (Upper) An
aliquot (1 mL) of the GPAO/DAPY reaction mixture in 0.1 M
potassium phosphate buffer, pH 7.0, was mixed with 2 mL of Kovacs’
reagent, incubated at 50 C for 30 min and finally cooled in an
ice-bath The absorption spectrum was recorded against a blank
con-taining water instead of the reaction mixture; (upper) reaction mixture
(lower) reaction mixture after removing protein by ultrafiltration For
experimental details see Materials and methods (Lower) A 1 mL
portion of 5 m M DDD was mixed with 2 mL of Kovacs’ reagent,
incubated at 50 C for 30 min and finally cooled in an ice bath.
Absorption spectra were then recorded against a blank containing
water instead of the dihydropyridine.
Trang 8Three peaks at elution times 3.0 min (1), 15.8 min (2) and
23.0 min (3) were collected and their composition analyzed
using ESI-MS and MALDI-MS The largest peak
absorb-ing at 310 nm (peak 1) appeared to correspond to a sabsorb-ingle
chemical compound (m/z 178.1) The corresponding
ESI-IT-MS/MS spectrum is presented in Fig 6, where
fragmentation peaks were observed with m/z 161.1, 149.1, 144.1, 135.1, 132.1, 120.1, 109.1, 95.0 and 82.0 After lyophilization, a solid obtained from peak 1 did not produce irreversible inhibition of the studied enzymes There was one more compound in peak 2 with m/z 257.1, whose MS/MS spectrum provided peaks with m/z 240.1, 228.1, 214.3, 202.3, 176.2, 161.2, 149.2 and 133.0 Finally, peak 3 contained at least five compounds In addition to those with m/z 178.1 and 257.1 there were three more peaks with m/z 334.3 (fragmentation: m/z 317.3, 305.2, 291.1, 253.2 and 240.1), 350.2 (fragmentation: m/z 333.2 and 307.1) and 431.3 (fragmentation: m/z 414.3, 388.3 and 337.1) MALDI-TOF-MS of the separated peak 1 provided a single compound with m/z 178.1; the same m/z value was obtained by an ionization without using the HABA matrix MALDI-PSD-TOF-MS provided a fragmentation pattern consistent with the ESI-IT-MS/MS experiments already mentioned (data not shown)
ESI-Q-TOF-MS analysis of the HPLC peak 1 permitted the determination of both exact mass and elemental composition of the DAPY oxidation product An m/z value of 178.14 was obtained, which matches a molecular formula C10H16N3 Peaks in the corresponding MS/MS spectrum provided the following m/z values and elemental composition of ions: 161.11 (C10H13N2), 149.11 (C9H13N2), 135.09 (C8H11N2), 109.08 (C6H9N2), 95.06 (C5H7N2) and 82.06 (CHN)
Fig 5 ESI-IT-MS analyses of GPAO/DAPY
reaction mixtures (Upper) DAPY (5 m M ) in
0.1 M ammonium bicarbonate, pH 7.8, was
mixed with an excess of GPAO and incubated
at 30 C for 24 h After removing protein by
ultrafiltration, the reaction mixture was
ana-lyzed by ESI-IT-MS as described in Materials
and methods (Lower) A combined solution of
DAPY and ACA (each 5 m M ) in 0.1 M
ammonium bicarbonate, pH 7.8, was mixed
with an excess of GPAO and incubated at
30 C for 24 h After removing protein by
ultrafiltration, the reaction mixture was
ana-lyzed by ESI-IT-MS as described in Materials
and methods.
Fig 6 MS/MS spectrum of DAPY oxidation product The isolated
DAPY oxidation product from enzymatic microscale production was
dissolved in 0.3% (v/v) triethylamine acetate, pH 7.0, and separated by
RP-HPLC as described in Materials and methods The 310 nm-peak
at an elution time 3.0 min was collected and analyzed by ESI-IT-MS
and MS/MS The spectrum shown was recorded after
collision-induced fragmentation of the parent ion belonging to the DAPY
oxidation product (m/z 178.1).
Trang 9NMR spectroscopy of DAPY oxidation product
For initial experiments, DAPY oxidation by GPAO was
performed in 20 mMpotassium phosphate buffer made in
D2O (pD 7.0) The enzyme protein was removed by
ultrafiltration and the filtrate was directly measured Several
signals with the following chemical shifts were observed in
the13C-NMR spectrum: d (p.p.m.) 17.0, 25.1, 29.1, 37.9,
62.5, 72.1, 74.3, 82.7, 123.0, 131.2 and 159.8 The
corres-ponding 1H-NMR spectrum contained various signals in
the region 2.0–4.5 p.p.m., but these were largely obscured
by the residual water peak (4.5–5.0 p.p.m.) In addition, two
vinylic signals at d 5.7 and 7.8 were observed, but their
intensities were too small to ascertain their multiplicities
(not shown) Much better1H-NMR spectra were obtained
after the extraction of the ultrafiltered and lyophilized
GPAO/DAPY reaction mixture by CD3OD Figure 7
shows such a spectrum, including the following signals: d
2.63–2.73 (m), 3.12 (q, J¼ 6.95 Hz), 3.31 (p, J ¼ 1.65 Hz),
3.79 (t, J¼ 2.20 Hz), 4.19 (t, J ¼ 2.20 Hz), 5.35 (d, J ¼
6.22 Hz), 7.82 (d, J¼ 6.22 Hz) The spectrum is partially
obscured by two signals of residual methanol at d 3.3 and
4.6–5.1 There are also three complex signals that are quite
difficult to interpret, which are centered at d values of 2.80,
3.55 and 3.65.13C-NMR spectra measured with the GPAO/
DAPY mixture in CD3OD resembled those recorded in
D2O, but the obtained quality was lower.1H-NMR spectra
were also recorded in D2O using the solid obtained by
lyophilization of the peak 1 from the HPLC separation
mentioned above However, NMR signal intensities were
insufficient due to the low concentration of compound In
addition, these spectra were obscured by two peaks of
residual triethylamine from the elution buffer at d 1.2 and
3.2 (data not shown)
Other analyses
Lysine residues in plant CAOs are possible targets for
covalent binding of reactive electrophilic aminoaldehydes
formed by the turnover of acetylenic diamine substrates [7–11] The cDNA of GPAO subunit (without the signal peptide) has been cloned and sequenced (D Kopecˇny´,
N Houba-He´rin, H G Faulhammer & M Sˇebela, unpublished results; EMBL/GenBank accession number AJ786401) The translated protein sequence is largely similar to those two published for PSAO [32,33] and comprises 38 lysines GPAO and PSAO have very similar peptide maps obtained by MALDI-MS experiments [34] PSAO protein sequence of Koyanagi et al [32], which is deposited under accession number JC7251 (NCBI Protein Databank) differs slightly from that of Tipping and McPherson [33], accession number Q43077, in that whereas the former sequence comprises 39 lysine residues, the latter has only 38 lysine residues, as calculated for the mature form of the protein Although native PSAO (a dimer) is thus expected to comprise 76–78 lysine residues, only 32 are solvent-accessible [8] We determined 38 accessible lysines per dimer in the native GPAO using a modified protocol with the TNBS reagent, and 36 lysines per dimer (average values from repeated measurements) after reaction with the DAPY
Quinone-staining experiments [29] with the DAPY-inac-tivated GPAO were positive and demonstrated that the topaquinone cofactor was not modified in a redox-inactive form (data not shown) Chromatofocusing experiments performed according to that with DABY-inactivated GPAO [8] revealed that the pI value of the DAPY-inactivated GPAO was not dramatically changed The native GPAO
is characterized by a pI of 7.2 [18] After the reaction with
an excess of DAPY, the enzyme sample comprised more species having isoelectric points of pI 6.8–7.5 (Fig 8) Therefore, the inactivation resulted in a heterogeneous mixture of differently charged protein molecules
Discussion
DAPY was synthesized as an analog of cadaverine (pentane-1,5-diamine), which is known as the best substrate
Fig 7 1 H-NMR spectrum of DAPY oxidation product DAPY (5 m M ) in 2 mL of 20 m M potassium phosphate buffer, pH 7.0, was mixed with an excess of GPAO (5 mg, added as a concentrated solution in the same buffer) and the mixture was incubated at 30 C for 12 h The same amount of GPAO was added again and the incubation proceeded for an additional 12 h The resulting solution was centrifuged to remove protein precipitate and ultrafiltered, and the filtrate was lyophilized The NMR sample was finally prepared by extracting the lyophilizate with 0.5 mL of CD 3 OD The insets shows a detailed view of the vinylic doublet signals belonging to 2,3-dihydropyridine.
Trang 10of plant CAOs [4] Contrary to naturally occurring
diam-ines, the DAPY molecule contains a triple bond at the
b- and c-positions from the two primary amine termini The
oxidative conversion of the compound by GPAO and
OVAO was demonstrated by measuring the production of
H2O2using spectrophotometry Therefore, the enzymes are
able to undergo complete turnover [3] However, DAPY
was oxidized more efficiently by OVAO than by GPAO
Although, to date, OVAO has not been crystallized nor had
its structure solved, this observation might be explained in
terms of different arrangements of the active sites of the
enzymes resulting in the preference for C5-diamine
sub-strates by OVAO
Similarly to the conversion of its lower homolog DABY
by PSAO [7], DAPY oxidation by the studied enzymes led
to their irreversible inhibition The apparent inactivation
constants KIof 10)5Mare on the same order of magnitude
as those KI values previously described for BEA [6] and
DABY [7] in the reactions with lentil seedling amine oxidase
(LSAO) and PSAO, respectively The obtained rates of
inactivation resembled for example that for BEA as
measured with LSAO [6], but they were lower than that
for DABY in the reaction with PSAO [7] At the same time,
whereas the determined partition ratio values with GPAO
and OVAO were in the range observed for BEA (r¼ 100)
and some other monohalogenated alkylamines (r < 500) in
the reactions with LSAO [6], they were significantly higher
than that for DABY and PSAO (r¼ 17) [7] From this
point of view, plant CAOs are more resistant to the
inactivation by DAPY than by DABY Binding of DAPY
at the active site of GPAO is dependent on both pH and
ionic strength In these features, the reaction does not differ
from those of typical plant CAO substrates like putrescine
or cadaverine GPAO and OVAO inactivation caused by
the substrate DAPY fulfills the criteria of a
mechanism-based inhibition: it is time dependent, irreversible and can be
weakened in the presence of a normal substrate [5,7,11,22]
DAPY oxidations by GPAO and OVAO were
accom-panied by spectral changes The typical absorption
spec-trum of a substrate-reduced CAO, which appeared after the
rapid addition of DAPY to either GPAO or OVAO
solutions, was in accordance with the substrate properties of
DAPY determined by the guaiacol spectrophotometric
assay Presuming that DAPY oxidation follows the same mechanism as for common substrates of plant CAOs, the reaction should generate 5-amino-2-pentynal or 5-amino-3-pentynal as a product aldehyde (DAPY is not a symmetric molecule) Although no free aldehyde was detected by TLC
in the reaction mixture, indirect evidence for an amino-pentynal turnover product was that an adduct formed (m/z 211.3) when DAPY was enzymatically oxidized in the presence of ACA This adduct exhibited a MS/MS fragmentation pattern similar to that of free ACA, showing
a loss of a water molecule from the carboxylic group ()18, m/z 211.3 fi m/z 193.3)
There are detailed reports on the mechanism-based inhibition of CAOs by DABY in the literature [7–11] The authors have shown that DABY is oxidized to 4-amino-2-butynal, which induces inactivation by adducting to a nucleophile in the substrate channel In addition, the reaction brings about multiple surface labeling of the enzyme, which probably occurs through solvent-accessible nucleophilic residues [8] DAPY oxidation appears to result
in much less extensive protein modification, as the number
of free primary amino groups in the enzyme did not change dramatically Chromatofocusing of DAPY-inactivated GPAO revealed only a small change in the isoelectric point, likely caused by the modification of a few amino acid residues upon binding of aminopentynal This binding seems to be nonspecific, as the existence of some micro-heterogeneity (at least two species with different pI values)
in the inactivated enzyme was confirmed As in the case of DABY, the cofactor topaquinone is not modified by the reaction, as demonstrated by an unchanged quinone redox staining
Absorption spectroscopy demonstrated the formation of
a secondary product in the GPAO/DAPY reaction mixture with kmax at 310 nm and emitted fluorescence (kmax at
445 nm) upon excitation at 310 nm, supportive of extended conjugation Dialysis of the reaction mixtures containing GPAO or OVAO and DAPY, resulted in decoloration, demonstrating that the chromophore generated is a free low molecular mass compound Several colorimetric assays provided evidence for the presence of a nitrogenous heterocycle, in addition to a free amino group The GPAO/DAPY reaction mixture exhibited a positive reac-tion with ABA and ninhydrin reagents, similar to that observed for the cyclic imines 1-pyrroline and 1-piperideine formed upon enzymative oxidation of putrescine and cadaverine, respectively In acidic medium, DMAB and DMAC reacted with the DAPY oxidation product (and also with the model compounds DDD and NADH) to give markedly colored adducts This probably occurs upon binding of the reagents at the a-position of the heterocycle [8]
The rigidity of the triple bond in the presumed amino-pentynal turnover product would prevent cyclization, but this geometrical constraint would be relaxed by conjugate addition of a nucleophile Thus, as shown in Fig 9, if a molecule of unreacted DAPY is added to either of the two possible aminopentynal turnover products, cyclization to a six-membered heterocycle and eventual formation of a common resonance-stabilized 4-amino-2,3-dihydropyridine would be predicted The extended conjugation would be consistent with the observed absorption and fluorescence
Fig 8 Chromatofocusing of DAPY-inactivated GPAO
Chromatofo-cusing was performed on a Mono P HR 5/20 column using a BioLogic
Duo Flow liquid chromatograph at a flow rate of 1 mLÆmin)1 The
loading buffer was 25 m M Tris/HCl, pH 8.2, and the elution buffer was
a diluted mixture of Polybuffer 96 and Polybuffer 74 adjusted to
pH 5.0 with acetic acid All samples were dialyzed against the loading
buffer before separation Approximately 5 mg of protein was loaded.