Supramolecular protein-protein complexation via specific interaction between glycosylated myoglobin and sugar-binding protein

Article abstract of DOI:10.1080/10610270903254175

Two different types of artificial glycosylated haemins (glycohaemins), in which a monosaccharide (galactose) or a disaccharide (lactobionic acid, 4-O-β-galactopyranosyl-D-gluconic acid) was introduced at the terminals of the two haempropionate side chains, were synthesised to serve as a designed interface on the myoglobin surface. These glycohaemins were successfully inserted into apomyoglobin to yield an artificial glycoprotein by the conventional method. The interprotein interaction between the reconstituted myoglobin and peanut agglutinin lectin (PNA), a β-galactose-recognising protein, was confirmed by two different assay systems, i.e. a fluorometric assay using the fluorescein isothiocyanate-labelled lectin and an ELISA-like assay using the peroxidase-labelled lectin. The results revealed that each myoglobin reconstituted with the glycohaemin makes a complex with PNA, in which the glycoprotein with the disaccharides showed a higher binding affinity with the lectin compared to the glycoprotein with the monosaccharides, suggesting that the binding property clearly depends on the deposited carbohydrate surface on the myoglobin.

Full text of DOI:10.1080/10610270903254175

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Supramolecular protein–protein complexation via
specific interaction between glycosylated myoglobin
and sugar-binding protein
Hirokazu Nagai a , Akira Onoda a b , Takashi Matsuo a & Takashi Hayashi a
a Department of Applied Chemistry, Graduate School of Engineering , Osaka University ,
Suita, Osaka, 565-0871, Japan
b Frontier Research Base for Global Young Researchers, Graduate School of Engineering ,
Osaka University , Suita, Osaka, 565-0871, Japan
Published online: 02 Dec 2009.
To cite this article: Hirokazu Nagai , Akira Onoda , Takashi Matsuo & Takashi Hayashi (2010) Supramolecular protein–protein
complexation via specific interaction between glycosylated myoglobin and sugar-binding protein, Supramolecular Chemistry,
22:1, 57-64, DOI: 10.1080/10610270903254175
To link to this article: http://dx.doi.org/10.1080/10610270903254175
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Supramolecular Chemistry
Vol. 22, No. 1, January 2010, 57–64
Supramolecular protein–protein complexation via specific interaction between glycosylated
myoglobin and sugar-binding protein
Hirokazu Nagaia, Akira Onodaab, Takashi Matsuoa and Takashi Hayashia*
b
aDepartment of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan; Frontier
Research Base for Global Young Researchers, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
(Received 18 May 2009; final version received 4 August 2009)
Two different types of artificial glycosylated haemins (glycohaemins), in which a monosaccharide (galactose) or a
disaccharide (lactobionic acid, 4-O-b-galactopyranosyl-D-gluconic acid) was introduced at the terminals of the two haem-
propionate side chains, were synthesised to serve as a designed interface on the myoglobin surface. These glycohaemins
were successfully inserted into apomyoglobin to yield an artificial glycoprotein by the conventional method. The
interprotein interaction between the reconstituted myoglobin and peanut agglutinin lectin (PNA), a b-galactose-recognising
protein, was confirmed by two different assay systems, i.e. a fluorometric assay using the fluorescein isothiocyanate-labelled
lectin and an ELISA-like assay using the peroxidase-labelled lectin. The results revealed that each myoglobin reconstituted
with the glycohaemin makes a complex with PNA, in which the glycoprotein with the disaccharides showed a higher binding
affinity with the lectin compared to the glycoprotein with the monosaccharides, suggesting that the binding property clearly
depends on the deposited carbohydrate surface on the myoglobin.
Keywords: protein–protein complex; haemoprotein modification; haem-substitution; artificial glycoprotein
Introduction
to form the carbohydrate-linked amino acid residues.
In contrast, we have recently proposed a new method
which is an introduction of carbohydrate moieties into a
peripheral side chain of an enzyme cofactor (12). For
example, several haemoproteins will be suitable for
introducing a carbohydrate cluster onto the protein surface
because it is well known that protohaem IX in myoglobin
is readily replaced by an artificial haemin having a
functional group at the haem-propionate side chains
(13–15). In fact, we have already reported that the
negatively charged myoglobin reconstituted with the
artificially created haemin with a carboxylated cluster
bound to the two haem-propionate side chains serves as
a complex with the positively charged cytochrome c
(16, 17).
Biological systems have created sophisticated supramole-
cular assemblies of biomolecules, including proteins,
nucleic acids, lipids and sugars, which enable them to
integrate their various functions. To mimic and/or modify
the naturally occurring supramolecular assemblies using
an artificial methodology is one of the current interests
from the view point of constructing nanobiomaterials
(1–5). Especially, a cluster of carbohydrate molecules
serves as a crucial recognition interface mediating
protein–protein or protein–membrane interactions,
which triggers cellular communications, such as cell
adhesion, fertilisation, bacterial/viral infection and cancer
metastasis (6–8). Therefore, carbohydrate molecules are
attractive candidates for an interface displayed on a protein
to construct an artificial supramolecular architecture.
During the past decade, many efforts have been
devoted to developing at least three strategies producing
artificially glycosylated proteins as a surrogate for post-
translational modification (9–11): (i) preparation of a
small glycoprotein by solid phase peptide synthesis using a
glycosylated amino acid, (ii) introduction of carbohydrate
moieties into a terminal of the residue of reactive amino
acids such as Lys, Cys or Asp on the protein surface and
(iii) expression of proteins using a genetically coded
unnatural RNA with an amino acid having carbohydrate
derivatives. These methods involve modification in order
In our previous paper, we reported the preparation of
myoglobin modified with tetradentate galactose units
which has a sufficient binding affinity with peanut
agglutinin lectin (PNA), a b-galactose binding protein,
determined by a qualitative binding analysis (12). PNA is
known to recognise the monosaccharide moiety and more
strongly bind to the disaccharide; the interaction between
the carbohydrate moiety and lectin depends on the
structures and glycosylation pattern of the carbohydrates
(18). To investigate the expandability of our reconstitu-
tional strategy in constructing artificial glycoproteins, we
now describe the preparation of myoglobins reconstituted
*Corresponding author. Email: thayashi@chem.eng.osaka-u.ac.jp
ISSN 1061-0278 print/ISSN 1029-0478 online
q 2010 Taylor & Francis
DOI: 10.1080/10610270903254175
http://www.informaworld.com

58
H. Nagai et al.
with glycosylated haemins (glycohaemins) containing a
monosaccharide unit, galactose (Gal) and a disaccharide
unit, lactobionic acid (GlcGal) (Figure 1), as well as their
sufficient binding abilities with the target lectin.
determined on a Synergy HT SIAFR-4 (BIO-TEK
Instruments, Inc., Winooski, VT, USA) at 470 nm.
Fluorescent spectra were recorded using HITACHI
F-4500. Distilled water was demineralised by a Barnstead
NANOpure DIamondTM apparatures.
Experimental
Chemicals
Instruments
All reagents and chemicals were obtained from commer-
cial sources and used without further purification unless
otherwise noted. Glycohaemins 1 and 2 were synthesised
by the methods described below. Native horse heart
myoglobin (nMb) was purchased from Sigma Co., Ltd
(St Louis, MO, USA; M-1882). Bovine serum albumin
(BSA) and horseradish peroxidase-labelled peanut
agglutinin lectin (HRP-PNA) (L7759) were purchased
from Sigma Co., Ltd. Fluorescein isothiocyanate-labelled
peanut agglutinin lectin (FITC-PNA) was purchased from
Seikagaku Corporation (Tokyo, Japan).
1H NMR spectra were recorded on a Bruker AVANCE 400
(400 MHz) and/or JEOL JNM-EX 270 (270 MHz)
spectrometers. The 1H NMR chemical shifts were reported
in ppm relative to the residual solvent resonances. Mass
analysis was carried out using a time-of-flight mass
spectrometer (TOF MS) equipped with electrospray
ionisation on an Applied Biosystems Mariner API-TOF
Workstation. UV–vis spectra were recorded using a
Shimadzu UV-2550 double-beam spectrometer. Gel
permeation chromatography (GPC) was performed on a
LC-908 recycling preparative HPLC system (Japan
Analytical Industry Co., Ltd, Tokyo, Japan) connected in
series with JAIGEL-1H, JAIGEL-2H and JAIGEL-3H
columns (Japan Analytical Industry Co., Ltd) using CHCl3
as an eluent. Preparative high-performance liquid
chromatography (HPLC) was performed by a Shimadzu
SCL 10 Avp HPLC system. Purification of the
reconstituted myoglobins (rMbs) was performed by an
Synthetic procedures
General
Protoporphyrin IX having four carboxylic acid groups
(3) was synthesised according to our previous paper (12).
2-( p-Nitrobenzyloxycarbonylamino)ethylamine (19) and
2-(benzyloxycarbonylamino)ethylamine (20) were pre-
pared according to the procedures reported in the
literature.
¨
Amersham Bioscience AKTAFPLC system equipped with a
Superdex 75 column and a fraction collector Frac-920 at
48C. Optical absorptions for the microplate assays were
Figure 1. Schematic representation of the reconstitution strategy of myoglobin with synthetic haemin (glycohaemin).

Supramolecular Chemistry
59
Synthesis of 2-(p-nitrobenzyloxycarbonylamino)ethyl
2,3,4,6-tetra-O-acetyl-b-D-galactopyranoside (4)
subjected to silica gel chromatography (CHCl3/
MeOH ¼ 9/1) and recycle GPC to afford purple solid 8
(124mg, 27%). ESI-TOF MS (positive mode) m/z 887.82
(Mþ3H)3þ, calcd for (C124H173N12O52)3þ 887.37; UV–vis
(CHCl3) lmax (nm) 403, 504, 538, 574, 628 and 664.
To a solution of 2-( p-nitrobenzyloxycarbonylamino)ethyl-
amine (2.7 g, 11.2 mmol) and 1,2,3,4,6-tetra-O-acetyl-b-
D-galactopyranose (3.9 g, 10 mmol) in CH2Cl2 (90 ml) was
added dropwise to a solution of boron trifluoride
diethyletherate (9.0 ml, 71 mmol) at 08C. The reaction
mixture was stirred at 08C for 30 min, followed by at room
temperature for 12 h. The solution was neutralised by
NaHCO3 aq., and the organic phase was washed with H2O
and brine. The organic phase was dried over Na2SO4 and
evaporated to dryness. The residue was purified by silica
gel chromatography (hexane/AcOEt ¼ 1/4) to give 4
Synthesis of glycohaemin 1
To a solution of 8 (124 mg, 46 mmol) and suspended
NaHCO3 (386 mg, 0.46 mmol) in 6 ml of N2-purged
CHCl3/MeCN (1/1, v/v) was added ferrous chloride
tetrahydrate (92 mg, 0.46 mmol). The reaction mixture was
stirred at 508C for 2 h and cooled to room temperature. The
mixture was exposed to air and diluted with CHCl3 and
washed with water, sat. NaHCO3 aq. and brine. The
organic phase was dried over Na2SO4 and evaporated to
dryness. Half of the obtained residue was dissolved in
MeOH (5 ml) and NaOMe (5.5 mg, 100 mmol) was added.
The mixture was stirred at room temperature for 4 h and
100 ml of 0.1 M HCl aq. was added. After the solvent was
evaporated, the resulting residue was subjected to LH-20
gel filtration with elution of water to give glycohaemin 1
(31 mg, 64%). ESI-TOF MS (positive mode) m/z 1021.34
(MþHþ–Cl)2þ calcd for (C92H137FeN12O36)2þ1020.93;
UV–vis (100 mM posphate buffer) lmax (nm) 397 (sh)
and 596.
1
(1.7 g, 30%). H NMR (270 MHz, CDCl3) d (ppm) 8.21
(2H, d, J ¼ 8.9 Hz), 7.50 (2H, d, J ¼ 8.9 Hz), 5.38 (1H, d,
J ¼ 2.4 Hz), 5.26–5.14 (4H, m), 5.00 (1H, dd, J ¼ 3.5,
10.7 Hz), 4.46 (1H, d, J ¼ 7.8 Hz), 4.19–4.06 (2H, m),
3.92–3.84 (2H, m), 3.72–3.65 (1H, m), 3.44–3.37 (2H,
m), 2.17–1.85 (12H, m).
Synthesis of 2-aminoethyl 2,3,4,6-tetra-O-acetyl-b-D-
galactopyranoside (5)
To a solution of 4 (1.7 g, 3.0 mmol) in MeOH (30 ml) was
added 10% Pd/C (170 mg). After addition of AcOH (0.36 g,
6.0 mmol), the reaction mixture was stirred at room
temperature under H2 atmosphere for over 2 h. The Pd/C
was removed by filtration, and then the filtrate was
evaporated to dryness. The residue was lyophilised for
2 days to remove p-toluidine. The resulting residue was
dissolved in CH2Cl2 (20 ml), and p-toluenesulphonic acid
monohydrate (570 mg, 3.0 mmol) was added. The solution
was stirred at room temperature for 1.5 h. The solvent was
evaporated and co-evaporated with toluene three times to
give precipitate 5 as a tosylate salt (418 mg, 25%). 1H NMR
(270 MHz, CDCl3) d (ppm) 7.71 (2H, d, J ¼ 8.1 Hz), 7.21
(2H, d, J ¼ 8.1 Hz), 5.34 (1H, d, J ¼ 2.7 Hz), 5.15–5.08
(1H, m), 4.94 (1H, dd, J ¼ 3.2, 10.5 Hz), 4.48 (1H, d,
J ¼ 8.1 Hz), 4.19–4.07 (2H, m), 4.04–3.99 (2H, m), 3.88–
3.83 (1H, m), 3.30–3.15 (2H, m), 2.29–1.90 (15H, m).
Synthesis of 2-(benzyloxycarbonylamino)ethyl
(2,3,5,6,20,30,40,60-octa-O-acetyl)lactobionamide (6)
To a suspension of lactobionic acid (7.5 g, 21 mmol) in
MeOH (200 ml) was added 2-(benzyloxycarbonyl-
amino)ethylamine (4.1 g, 21 mmol). The reaction mixture
was stirred at 758C for 6 h. The solvent was evaporated to
give a semi-solid compound. The obtained residue was
dissolved in pyridine (270 ml) and cooled with ice bath.
Acetic anhydride (170 ml) was added dropwise at 08C
and the reaction mixture was stirred at room temperature
for 12 h. The reaction was quenched by addition of MeOH
(170 ml) at 08C and the solvent was removed by
evaporation. The residue was dissolved in AcOEt and
the organic phase was washed with water, 0.1 M HCl
aq., sat. NaHCO3 aq. and brine. The organic phase was
dried over Na2SO4 and evaporated to give crude 9. The
residue was purified by silica gel chromatography
(hexane/AcOEt ¼ 1/3–AcOEt only) to give white solid
6 (7.8 g, 43%). 1H NMR (400 MHz, CDCl3) d (ppm) 7.33–
7.24 (5H, m), 6.80 (1H, br), 5.61 (1H, d, J ¼ 5.9 Hz), 5.49
(1H, dd, J ¼ 3.5, 6.0 Hz), 5.35 (1H, d, J ¼ 3.0 Hz), 5.30
(1H, br), 5.16 (1H, dd, J ¼ 8.1, 10.6 Hz), 5.10–5.02 (3H,
m), 4.98 (1H, dd, J ¼ 3.5, 10.4 Hz), 4.62 (1H, d,
J ¼ 8.1 Hz), 4.52 (1H, dd, J ¼ 2.7, 12.4 Hz), 4.38 (1H,
br), 4.18–4.14 (1H, m), 4.04–3.98 (2H, m), 3.87 (1H, t,
J ¼ 6.6 Hz), 3.45–3.20 (4H, m), 2.17 (3H, s), 2.12 (3H, s),
2.05 (6H, s), 2.03 (3H, s), 2.00 (6H, s), 1.96 (3H, s).
Synthesis of porphyrin having peracetylated galactoside
(8)
To a solution of 5 (418mg, 0.74mmol), 3 (126mg,
0.17 mmol) and 1-hydroxybenzotriazole monohydrate
(114mg, 0.74mmol) in dry DMF (6 ml) was slowly added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (335mg, 1.75mmol) at 08C. The reaction mixture
was stirred at 08C for 1 h, followed by at room temperature
for 12h. The solution was diluted with CHCl3 and the
organic phase was washed with water, 5% citric acid, sat.
NaHCO3 aq. and brine. The organic phase was dried over
Na2SO4 and evaporated to give a purple solid. The solid was

60
H. Nagai et al.
purified by reverse phase HPLC (YMC-pack C18,
Synthesis of 2-aminoethyl (2,3,5,6,20,30,40,60-octa-O-
acetyl)lactobionamide (7)
250 £ 20 mm) to give glycohaemin 2 (10 mg, 36%). The
HPLC condition was as follows: CH3CN/H2O (both
containing 0.1% TFA) 10/90–50/50 (linear gradient over
50 min), flow rate ¼ 5.0 ml/min, detected by UV (400 nm).
ESI-TOF MS (positive mode) m/z (MþHþ –Cl)2þ
1375.44 calcd for (C116H181FeN16O56)2þ 1375.06; UV–
vis (H2O) lmax (nm) 358 (sh), 397 and 598.
To a solution of 6 (3.4 g, 3.9 mmol) in AcOH (40 ml) was
added 10% Pd/C (200 mg). The reaction mixture was
stirred at room temperature under H2 atmosphere over 12 h.
The Pd/C was removed by filtration, and then the filtrate
was evaporated to dryness. The resulting mixture was
dissolved in CH2Cl2 (20 ml), and p-toluenesulphonic acid
monohydrate (740 mg, 3.9 mmol) was added. The solution
was stirred at room temperature for 1.5 h. The solvent was
evaporated and co-evaporated with toluene three times to
give white solid 9 as a tosylate salt (3.4 g, 96%). 1H NMR
(400 MHz, CDCl3) d (ppm) 8.03 (1H, br), 7.69 (2H, d,
J ¼ 7.8 Hz), 7.51 (3H, br), 7.17 (2H, d, J ¼ 7.8 Hz), 5.50
(1H, br), 5.44 (1H, br), 5.34 (1H, d, J ¼ 3.2 Hz), 5.11–5.06
(2H, m), 4.97 (1H, dd, J ¼ 3.4, 10.5 Hz), 4.57 (1H, d,
J ¼ 7.8 Hz), 4.53 (1H, br), 4.28 (1H, br), 4.16 (1H, br),
4.08–3.96 (2H, m), 3.85 (1H, t, J ¼ 6.5 Hz), 3.48 (2H, br),
3.16 (2H, br), 2.11–1.94 (27H, m).
Reconstitution of myoglobin with glycohaemin
The nMb was dissolved in 10mM phosphate buffer (pH 6.0)
and purified by cation exchange chromatography using a
CM-52 cellulose column as previously described (13). The
horse heart apomyoglobin (apoMb) was prepared from
purified nMb by Teale’s acid-2-butanone method (21),
lyophilised and then stored at 2808C before use. To a
solution of apoMb in 100 mM phosphate buffer (pH 7.0)
was added dropwise an aqueous solution of 1.2 equivalent of
glycohaemin 1 with gentle shaking, followed by overnight
incubation at 48C. The rMb with glycohaemin 1, rMb–Gal,
was purified from the residual glycohaemin 1 using a
Superdex 75 column (10 mM phosphate buffer containing
154 mM sodium chloride as an eluent) in the FPLC system.
The rMb with glycohaemin 2, rMb–GlcGal, was obtained
according to a similar procedure. The approximate yield for
inserting the glycohaemin into apoMb was 40–50%.
Synthesis of porphyrin having peracetylated
lactobionamide (9)
To a solution of 3 (200mg, 0.17mmol), 9 (2.5g, 2.72mmol)
and triethylamine (2ml, 14mmol) in DMF (7ml) was added
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholi-
nium chloride (1.75 g, 6.3 mmol). The reaction mixture was
stirred at room temperature for 12h. The solvent was
evaporated and the residue was diluted with CH2Cl2. The
organic phase was washed with water, 0.1 M HCl aq., sat.
NaHCO3 aq. and brine. The organic phase was dried over
Na2SO4 and evaporated to give crude 9. The residue was
purified by silica gel chromatography (CHCl3/
MeOH ¼ 9/1–6/1) to give a purple solid. The solid was
further purified by recycle GPC to give 9 (200mg, 32%).
ESI-TOF MS (positive mode) m/z (Mþ3Hþ)3þ 1348.74
calcd for (C180H249N16O88)3þ 1347.52; UV–vis (CHCl3)
Fluorescent quenching assay
The fluorescence spectra were measured in a quartz
microcell at 258C and recorded from 500 to 600 nm with
excitation at 490 nm. Aliquots (150 mM, 2 ml) of the stock
solution of nMb, rMb–Gal or rMb–GlcGal were added to
a solution of FITC-PNA (43 mM, 60 ml). A 10 mM Tris–
HCl buffer (pH 7.0) containing 10 mM MnCl2 and CaCl2
was used. The changes in the relative FITC intensity at the
fluorescence maxima (520 nm) were plotted versus the
myoglobin concentration with the Stern–Volmer analysis,
lmax (nm) 408, 505, 541, 574, 630 and 668.
F0
Synthesis of glycohaemin 2
¼ 1 þ KS½Qꢀ;
F
To a solution of 9 (40 mg, 9.9 mmol) and suspended
NaHCO3 (15 mg, 0.18 mmol) in 4 ml of N2-purged
CHCl3/MeCN (1/1, v/v) was added ferrous chloride
hydrate (40 mg, 0.20 mmol). The reaction mixture was
stirred at 708C for 2 h and cooled to room temperature. The
mixture was exposed to air and diluted with CHCl3 and
washed with 1 M HCl aq. and brine. The organic phase was
dried over Na2SO4 and evaporated. The obtained residue
was dissolved in 2 ml of MeOH/H2O (1/1, v/v) and
NaOMe (5.5 mg, 100 mmol) was added. After the reaction
mixture was stirred at room temperature for 5 h, amberlite
IRC-50 was added. The IRC-50 was removed by filtration
and the solvent was evaporated. The resulting residue was
where F0 and F are the fluorescence intensity in the absence
and presence of the quencher (rMb–GlcGal, rMb–Gal and
nMb), respectively. KS represents the association constant
and [Q] is the concentration of the quencher (22).
Microplate assay
The commercially available Nunc Maxisorp F96 microwell
plates were used for this assay and the procedures are
described below. The wells were coated with a solution of
nMb, rMb–Gal or rMb–GlcGal (2 mM, 100 ml). The plate

Supramolecular Chemistry
61
was incubated overnight at room temperature to immobilise
the protein on the well surface. The protein solution was
removed, and the wells were washed three times with
phosphate buffered saline (PBS; 250 ml), to remove any
unbound proteins. The redundant well surface was blocked
by the addition of 1% BSA in PBS (100ml), with incubation
for 1 h so as to avoid the non-specific binding of the PNAs to
the well. The blocking solution was removed, and the wells
were washed three times with PBS (250ml). HRP-PNA
(50 mg/ml, 100 ml)wasaddedandthe plate wasincubatedfor
2 h. The protein solution was removed and the wells were
washed three times with PBS (250ml). To each well were
added the substrate solution (guaiacol, 1 mM, 100 ml) and
H2O2 (1 mM, 100 ml). The increases in the 470 nm
absorbance, which corresponds to the oxidised product
catalysed by HRP, were observed on a microplate reader.
to the apoprotein, the Soret band of the glycohaemin shifted
from 397 to 408 nm, suggesting that the modified haemin
is incorporated into the myoglobin haem pocket and is
coordinated with His93 to afford the five-coordinate
Fe(III) species.
The rMbs were confirmed by UV–vis and ESI-TOF
MS spectroscopic methods and size exclusion chromato-
graphic analysis (SEC). The UV–vis absorption spectra of
rMbs showed the characteristic Soret absorptions at 408 nm
and the Q absorption at 506 and 633 nm similar to those of
nMb, indicating that the glycohaemins were incorporated
at the proper position with the same orientation in the haem
pocket of the myoglobin (Figure 2). The ESI-TOF MS
spectra of rMb with glycohaemin 1 (rMb–Gal) and rMb
with glycohaemin 2 (rMb–GlcGal) exhibited the corre-
sponding multiple charged ions of the holo- and/or apo-
proteins.1 The SEC traces of nMb (17.5 kDa), rMb–Gal
(19 kDa) and rMb–GlcGal (20 kDa) are in good agreement
with the relative increase in their molecular size/weight
because of the attached carbohydrate moiety anchored at
the terminal of the two haem-propionate side chains
(Figure 3).
Results and discussion
Preparation of glycohaemins
Two artificial haemins having tetradentate monosacchar-
ide (glycohaemin 1) or disaccharide (glycohaemin 2)
moieties were synthesised according to the synthetic route
shown in Scheme 1. Galactose was coupled with 2-( p-
nitrobenzyloxycarbonylamino)ethylamine and then the
protecting group was removed by hydrogenation to obtain
the galactose with the amino group, 5. We also prepared
the corresponding Z-protected galactose; however, the
removal of the Z group was unsuccessful under the general
hydrogenation conditions, whereas in the case of the
synthesis of 6, the removal of the Z group proceeded very
smoothly to give the disaccharide analogue with the amino
group, 7. These sugar moieties were coupled to the
modified protoporphyrin IX precursor 3 with bidentate
carboxyl linkers at the terminal of both propionate chains,
according to our previous method (12).
Fluorescence quenching assay
To evaluate the interaction between the carbohydrate
moieties attached on the myoglobin surface and PNA,
we carried out fluorescence quenching assay using the
FITC-PNA (23–25), in which the fluorescence can be
quenched by the iron-containing haemin group when the
FITC chromophore and the haemin are located within the
acceptable distance. Thus, the decay of the FITC
fluorescence intensity can serve as a useful probe to
estimate the complexation between the PNA and the sugar
moiety linked to the myoglobins.
The changes in the relative FITC intensity at the
fluorescence maxima (520 nm) were plotted versus the
myoglobin concentration as shown in Figure 4. In the case
of the nMb, the FITC fluorescence intensity slightly
decreased upon the addition of the myoglobin solution
because of non-specific interactions between PNA and
nMb. However, in the case of glycomyoglobins, rMb–Gal
and rMb–GlcGal, more significant decays in the
fluorescence intensity were observed with the increased
concentration of the reconstituted proteins, suggesting
specific binding between the attached carbohydrate groups
on the protein surface and PNA. Furthermore, the results
revealed that the terminal galactoside residue in the
disaccharide-conjugated myoglobin, rMb–GlcGal, exhi-
bits superior binding as compared with the monosacchar-
ide-conjugated myoglobin, rMb–Gal.
Iron insertion into the glycosylated protoporphyrins 8
and 10 was carried out under conventional conditions. The
glycohaemins 1 and 2 were obtained by removal of the
acetyl groups under the alkaline conditions, and purified
by gel filtration and/or reverse phase HPLC techniques.
The porphyrins and metalloporphyrins are generally
known to be aggregated and insoluble because of their
hydrophobic nature, while our glycohaemins with attached
multiple carbohydrate moieties exhibit a high solubility in
aqueous solutions at physiological pH (10 mM phosphate
buffer, pH 7.0) in which the glycohaemins existed as the
monomer and m-oxo dimer indicated by the characteristic
absorption at 605, 397 and 360 nm (sh).
The PNA forms homotetramer in which each domain
has one substrate binding site. The four binding sites in the
PNA are sufficiently separated by a distance of more than
Reconstitution of myoglobin with glycohaemin
The rMbs with glycohaemins were prepared by the
conventional method. Upon the addition of the glycohaemin
˚
50 A. The maximum distance between the sugar moieties

62
H. Nagai et al.
Scheme 1. Syntheses of glycohaemins 1 and 2. Reagents: (a) ( pNZ)HNCH2CH2OH, BF3·Et2O, CH2Cl2; (b) (i) H2, 10% Pd/C, MeOH,
AcOH; (ii) lyophilisation; (iii) p-toluenesulphonic acid monohydrate, CH2Cl2; (c) (i) ZHNCH2CH2NH2, MeOH; (ii) Ac2O, pyridine; (d)
(i) H2, 10% Pd/C, AcOH; (ii) p-toluenesulphonic acid monohydrate, CH2Cl2; (e) 5, HOBt·H2O, EDC·HCl, DMF; (f) (i) FeCl2, NaHCO3,
CHCl3/MeCN; (ii) NaOMe, MeOH; (g) 7, DMT-MM, DMF; (h) (i) FeCl2, NaHCO3, CHCl3/MeCN; (ii) NaOMe, MeOH/H2O.
˚
in the synthetic haemin is around 30 A, and hence it seems
rather difficult for one glycosylated Mb to occupy two of
the four binding sites in a bidentate fashion at the same
time. The Stern–Volmer plot showed a linear relationship
under diluted condition, indicating that the binding site is
independently occupied without any allosteric effect.
Using the fluorescence quenching results, the association
constant of the three Mbs with each domain of the PNA
was determined as follows: rMb–GlcGal (2.7 £ 104 M21),
rMb–Gal (1.3 £ 104 M21) and nMb (5.3 £ 103 M21).
In fact, it is known that the PNA binds the tumour-
associated T-antigenic disaccharide, Galb1, 3GalNAc,
more strongly than the monosaccharide, galactose (26). It
seems likely that the gluconic acid of lactobionic acid
additionally supports the binding event by making
hydrogen bonds with PNA. In our case, ‘glycomyoglobin’
clearly binds to PNA via specific carbohydrate–lectin
interactions, which are dependent on the structure of the
carbohydrate.
Figure 2. UV–vis spectra of rMb–GlcGal (dotted line), rMb–
Gal (dashed line) and nMb (solid line) in 10 mM phosphate buffer
at pH 7.0, 258C.
Figure 3. Size exclusion chromatographic profiles of rMb–
GlcGal (20 kDa; dotted line), rMb–Gal (19 kDa; dashed line) and
nMb (17.5 kDa; solid line).

Supramolecular Chemistry
63
Microplate assay
The above-mentioned results in the fluorescence quench-
ing experiments showed that our glycomyoglobins have
the potential to provide a useful binding interface for
sugar-binding proteins, such as lectins, in a homogeneous
solution. To demonstrate this strategy which is applicable
for other varieties of sugar moieties, high-throughput
screening methods would be desirable. Thus, we assessed
the binding ability of the rMbs to PNA in heterogeneous
circumstances using a microplate. The microplate assay is
similar to the well-known ELISA (11, 27–30), which has
Figure 5. Schematic representation of the principle of the
microplate assay technique.
already been demonstrated as a useful methodology for
high-throughput screening for biomolecular interactions,
such as protein–protein, protein–DNA and protein–small
molecule interactions. In our case, the reconstituted
protein was adsorbed on a microwell and treated with the
HRP-PNA conjugate as shown in Figure 5. Guaiacol,
2-methoxyphenol, was used as a substrate for the oxidation
catalysed by HRP-PNA, because it is catalytically
converted to the oxidised product which is determined
by spectral changes at 470 nm (31). The binding fashion
between PNA and glycomyoglobin was confirmed by a
time-course study of the increased absorption of the
oxidised guaiacol as shown in Figure 6. The profiles for
rMb–Gal and rMb–GlcGal revealed a significant increase
in the absorbance of the oxidised product. The native
myoglobin is assumed to bind to PNA non-specifically,
resulting in the slight increasing of the absorbance. The
reconstituted proteins accelerate the guaiacol oxidation,
suggesting that the introduction of the carbohydrate cluster
on the protein surface acts as an interface for PNA.
In particular, the faster guaiacol oxidation catalysed by
HPR-PNA in the presence of rMb–GlcGal demonstrates a
higher affinity of rMb–GlcGal for PNA compared to
rMb–Gal. Furthermore, it was found that the addition of
the excess amounts of galactose to the solution inhibited
the guaiacol oxidation, supporting the fact that the PNA
substrate binding sites selectively interact with the
carbohydrate cluster deposited on the myoglobin surface.
These results are in good agreement with the fluorescence
quenching assay, suggesting that the rMb immobilised on
the well surface provides the effective orientation of the
carbohydrate units as a recognising interface. Thus, we
found that the microplate assay is an appropriate method
for assessing the binding potential of the carbohydrate
Figure 4. (a) Fluorescent spectral changes for FITC excited at
490 nm upon the addition of rMb–GlcGal in 10 mM Tris–HCl
buffer (pH 7.0), [CaCl2] ¼ [MnCl2] ¼ 10 mM at 258C. The
concentration of protein is as follows: [rMb–GlcGal] ¼ 0, 4.8,
9.4, 14, 18, 21, 25, 28, 32, 35 and 38 mM and [FITC-PNA] ¼ 43,
42, 41, 39, 38, 37, 36, 35, 34, 33 and 32 mM. (b) Relative
fluorescent intensities (520 nm) versus titrated concentration of
myoglobins. rMb–GlcGal (filled circles), rMb–Gal (filled
squares) and nMb (filled triangles).

64
H. Nagai et al.
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Summary
We synthesised artificially modified haemins having a
monosaccharide unit, glycohaemin 1, and a disaccharide
unit, glycohaemin 2. These glycohaemins were smoothly
inserted into apoMb to give rMbs, which were well
characterised by UV–vis and ESI-TOF MS spectroscopies
and SEC analysis. The binding behaviour of rMb was
evaluated by two independent assays, suggesting that the
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Acknowledgements
We are grateful to Prof. Hiroshi Uyama (Osaka University) and Dr
Eiko Mochizuki (Osaka University) for their kind help in the
enzyme assays. This work was supported by MEXT and JSPS. One
of the authors, H.N., expresses his special thanks to the Global COE
(Center of Excellence) Program ‘Global Education and Research
Center for Bio-Environmental Chemistry’ of Osaka University.
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1. The apoprotein was formed under ionisation condition.