| Protein
& Peptide Letters
ISSN: 0929-8665
Protein & Peptide Letters
Volume 15, Number 8, 2008
Contents
Immobilized Biomolecules and Electronic Sensing
Devices
Guest Editor: Fakhri Saida
Editorial: Pp.
756
Biomolecule Immobilization in Biosensor Development: Tailored
Strategies Based on Affinity Interactions Pp.
757-763
B. Prieto-Simón, M. Campàs
and J.-L. Marty
[Abstract]
Protein-Based Voltammetric Biosensors
Fabricated with Nanomaterials Pp. 764-771
D. Zhang, J. Zhao and G. Li
[Abstract]
Reagentless Optical Biosensors for Organic
Compounds Based on Autoindicating Proteins Pp. 772-778
J. Galbán, V. Sanz, E. Mateos, I.
Sanz-Vicente, A. Delgado-Camón and S. de Marcos
[Abstract]
Propionate Sensor Using Coenzyme-A Transferase
and Acyl-CoA Oxidase Pp. 779-781
K. Sode, W. Tsugawa, M. Aoyagi, E. Rajashekhara
and K. Watanabe
[Abstract]
Label-Free Electrochemical Immunosensor
for the Determination of Fetoprotein Based on Core-Shell-Shell
Nanocomposite Particles Pp. 782-788
A.-L. Sun, Q.-A. Qi and Z.-L. Dong
[Abstract]
Development of an Electrochemical Biosensor
for the Rapid Detection of Naphthalene Acetic Acid in Fruits
by Using Air Stable Lipid Films with Incorporated Auxin-Binding
Protein 1 Receptor Pp. 789-794
D.P. Nikolelis, N. Ntanos, G.-P. Nikoleli
and K. Tampouris
[Abstract]
Cholinesterase Biosensor Construction
– A Review Pp. 795-798
M. Pohanka, D. Jun, H. Kalasz and K.
Kuca
[Abstract]
Aptabodies – New Type of Artificial
Receptors for Detection Proteins Pp. 799-805
T. Hianik, A. Porfireva, I. Grman and
G. Evtugyn
[Abstract]
General Articles
Regular Papers
Determination of Binding Potency of Peptidic Inhibitors of
Grb2 SH2 by Using the Protein-Captured Biosensor Method
Pp. 806-810
F.-D.T. Lung, W.-C. Li, Y.-H. Chang and
H.-M. Chen
[Abstract]
Circular Dichroism Reveals Sensitivity
of Glucagon Solution Structure to Fluoroalcohols, pH and Ionic
Strength Pp. 811-817
S. Guest, V.V. Ngo and M.A. Hefford
[Abstract]
Changes in Structure and in Interactions
of Heat-Treated Bovine β-Lactoglobulin
Pp. 818-825
S.H.-A. Mousavi, A.-K. Bordbar and
T. Haertlé
[Abstract]
Urea and Acid-Induced Unfolding of Fatted
and Defatted Human Serum Albumin Pp. 826-833
P. Salahuddin
[Abstract]
Classification of Amine Type G-Protein
Coupled Receptors with Feature Selection Pp.
834-842
Q.-B. Gao, C. Wu, X.-Q. Ma, J. Lu and
J. He
[Abstract]
Structural Bioinformatics Study of PNP
from Listeria monocytogenes Pp. 843-849
L.F.S.M. Timmers, R.A. Caceres, L.A. Basso,
D.S. Santos and W.F. De Azevedo, Jr.
[Abstract]
Telmisartan Inhibits Advanced Glycation
End Products (AGEs)- Elicited Endothelial Cell Injury by Suppressing
AGE Receptor (RAGE) Expression Via Peroxisome Proliferator-Activated
Receptor -γActivation
Pp. 850-853
S.-i. Yamagishi, T. Matsui, K. Nakamura,
M. Takeuchi and H. Inoue
[Abstract]
Preparation and Characterization of a
Novel Recombinant Human Parathyroid Hormone (1–34) Analog
(Gly1-Gln26-rhPTH(1
34)) with Enhanced Biological Activity Pp.
854-860
X.-C. Xu, S.-D. Zhong, F. Kai, L.-R. Li,
C. Liu, B. Liu and J.-K. Bao
[Abstract]
Antimicrobial and Anti-Inflammatory Activities
of Designed Antimicrobial Peptide P18 Analogues Pp.
861-865
Y.H. Nan and S.Y. Shin
[Abstract]
Abstracts
[Back to top]
Editorial:
Analyte detection by immobilized, biologically active macromolecules
(biomolecules) has emerged as a highly effective technology
serving the needs of both diagnostic industry and fundamental
proteomic research [1]. The vast majority of immobilized biomolecules
are proteins, very often enzymes or antibodies. Recently,
the immobilization of DNA and RNA molecules has added a new
array of applications to the biosensing technology [2].
Although important innovations have been made in the past
ten years in order to build reliable biosensors, important
challenges have yet to be met. Biomolecules immobilization
needs to be simple, inexpensive, highly repeatable (especially
in a manufacturing environment) and without interference with
the biological processes being measured. Biomolecules need
to sustain the harsh chemical treatments used during the immobilization
process. The immobilized biomolecules also need to be stable
(6 to 12 months at 4°C).
This is a particularly delicate task especially when the requirement
is to maintain the activity/the structural integrity of proteins
in an artificial, semi-solid, environment that very often
lacks adequate ionic strength, buffering power and anti-oxidant
capabilities.
Another key challenge encountered during the development of
reliable biosensors is the efficiency of communication between
the immobilized biomolecule (that serves as a receptor) and
the inorganic material in its vicinity that plays the role
of signal transducer. Tailored bio-interfaces and oriented
immobilization are some of the key solutions to this challenge
[3].
The selection of articles included in this special issue is
aimed at presenting the recent advances in biomolecules immobilization,
signal acquisition and signal processing. For this purpose,
an illustrative variety of protein-based and nucleic acid-based
sensors is described and discussed. A particular stress is
put on the performance of the sensing device in terms of selectivity
and sensitivity. The present special issue is composed of
three reviews and five original research articles.
The first review describes the main biomolecules immobilization
strategies with an emphasis on oriented immobilization techniques
(Prieto-Simon B. et al.). A second review (by Zhang
D. et al.) details the design of protein-based voltammetric
biosensors fabricated with nanomaterials. The third review
(by Galban J. et al.) is dedicated to the detection
of organic compounds using reagentless optical biosensors.
The research articles included in this issue describe the
development of new sensitive sensors for the detection of
propionate (Sode, K. et al.),α-1-fetoprotein
(Sun A-L. et al.), naphthalene acetic acid (Nikolelis
D.P. et al.) and organophosphates and carbamates
(Pohanka M. et al.)
An original article by Hianik T. and co-workers reports on
the development of biosensors based on aptabodies: artificial
receptors formed by capping DNA aptamers with specific protein
binding sites.
REFERENCES
[1] Choi, J.W., Oh, B.K., Kim, Y.K. and Min, J. (2008)
J. Microbiol Biotechnol., 17, 5-14.
[2] Lucarelli, F., Tombelli, S., Minunni, M., Marrazza, G.
and Mascini, M. (2008) Anal. Chim. Acta.,
609, 139-59.
[3] Yuan, W., Dong, H., Li, C.M., Cui, X., Yu, L., Lu, Z.
and Zhou, Q. (2007) Langmuir, 23,
13046-52.
Dr. Fakhri Saida
Guest Editor
Protein & Peptide Letters
University of California
San Diego
USA
Present address: General Atomics,
San Diego, USA
[Back to top]
Biomolecule Immobilization in Biosensor Development:
Tailored Strategies Based on Affinity Interactions
B. Prieto-Simón, M. Campàs
and J.-L. Marty
The exponential development of biosensors as powerful
analytical tools in the last four decades mainly relies on
the high sensitivity and selectivity offered when detecting
the target analyte. The transducer and the biological receptor
are the bases of the biosensor development. Nevertheless,
the bioreceptor immobilisation is also important, playing
a key role in the retention of the biological activity, and
thus affecting the sensitivity. Parameters such as shelf-life
and surface regeneration also depend on the biomolecule immobilisation.
Researchers are focusing their efforts towards random and
oriented immobilisation procedures. Adsorption, entrapment,
cross-linking and electrostatic interactions provide randomly
immobilised biomolecules, sometimes partially hindering their
biological activity. Covalent binding and affinity interactions
may enable oriented biomolecule immobilisations, providing
controlled, reproducible and highly active modified surfaces.
This paper reviews the main immobilisation strategies used
in the biosensors development, putting special emphasis on
our contribution to mild and oriented immobilisation techniques.
[Back to top]
Protein-Based Voltammetric Biosensors Fabricated with Nanomaterials
D. Zhang, J. Zhao and G. Li
Protein-based voltammetric biosensors are sensors based on
the electric communication between proteins and electrodes.
Recently, more and more nanomaterials are utilized to assist
the fabrication of such kind of biosensors. In this review,
we mainly detail the biosensors constructed with different
kinds of nanomaterials depending on their categories in the
past two years.
[Back to top]
Reagentless Optical Biosensors for Organic Compounds Based
on Autoindicating Proteins
J. Galbán, V. Sanz, E. Mateos, I.
Sanz-Vicente, A. Delgado-Camón and S. de Marcos
Optical reagentless biosensors are one of the most promising
alternatives for producing selective, sensitive and autonomous
sensors for real life applications. These devices are based
on the efficient use of the spectroscopic properties of bioreagents,
mainly proteins, as transducers; avoiding in this way the
use of chemical colorant/fluorophores which usually limit
sensors performance. In this paper a brief state of the art
of the bioreagents being used in biosensors as well as recent
alternatives are discussed. The advantages of flavoenzymes
and hemeproteins as the basis for reagentless biosensors are
particularly stressed.
[Back to top]
Propionate Sensor Using Coenzyme-A Transferase and Acyl-CoA
Oxidase
K. Sode, W. Tsugawa, M. Aoyagi, E. Rajashekhara
and K. Watanabe
We developed an amperometric propionate sensor using comprised
of two recombinant enzymes, propionate coenzyme A CoA transferase
from Clostridium propionicum and short-chain acyl-CoA
oxidase from Arabidopsis thaliana. Response current
increased linearly with increase in propionate concentration
from 10 μM
to 100 μM.
The detection limit was 10 μM
propionate.
[Back to top]
Label-Free Electrochemical Immunosensor for the Determination
of Fetoprotein Based on Core-Shell-Shell Nanocomposite Particles
A.-L. Sun, Q.-A. Qi and Z.-L. Dong
A new approach toward the development of advanced immunosensors
based on chemically functionalized core-shell-shell magnetic
nanocomposite particles, and the preparation, characteristics,
and measurement of relevant properties of the immunosensor
useful for the detection of α-1-fetoprotein
(AFP) in clinical immunoassays. The core-shell NiFe2O4/3-aminopropyltriethoxysilance
(APTES) (NiFe2O4@APTES)
was initially prepared by covalent conjugation, then gold
nanoparticles were adsorbed onto the surface of NiFe2O2@APTES,
and then anti-AFP molecules were conjugated on the gold nanoparticles.
The core-shell-shell nanocomposite particles not only had
the properties of magnetic nanoparticles, but also provided
a good biocompatibility for the immobilization of biomolecules.
The core-shell-shell nanostructure present good magnetic properties
to facilitate and modulate the way it was integrated into
a carbon paste. The analytical performance of the immunosensor
was investigated by using an electrochemical method. Under
optimal conditions, the resulting composite presents good
electrochemical response for the detection of AFP, and exhibits
wide linear range from 0.9 to 110 ng/mL AFP with a detection
limit of 0.5 ng/mL. Moreover, the proposed immunosensors were
used to analyze AFP in human serum specimens. Analytical results,
obtained for the clinical serum specimen by the developed
immunosensor, were in accordance with those assayed by the
standard ELISA. Importantly, the proposed immunoassay system
could be further developed for the immobilization of other
antigens or biocompounds.
[Back to top]
Development of an Electrochemical Biosensor for the Rapid
Detection of Naphthalene Acetic Acid in Fruits by Using Air
Stable Lipid Films with Incorporated Auxin-Binding Protein
1 Receptor
D.P. Nikolelis, N. Ntanos, G.-P. Nikoleli
and K. Tampouris
This work describes the investigations of electrochemical
interactions of naphthalene acetic acid (NAA) with stabilized
lipid films supported on a methacrylate polymer on a glass
fiber filter with incorporated auxin-binding protein 1 receptor
for the development of a biosensor for the rapid detection
of this compound in fruits. NAA was injected into the flowing
streams of a carrier electrolyte solution, the flow of the
electrolyte solution stops and an ion current transient was
obtained; the magnitude of the signal was correlated to NAA
concentration, which could be determined at the micromolar
level. NAA preconcentrates at the lipid membrane surface which
causes dynamic alterations of the electrostatic fields and
phase structure of membranes. The response times were ca.
5 min and naphthalene acetic acid was determined at concentration
levels of μM.
The effect of potent interferences included a wide range of
compounds. The results showed no interferences from these
compounds in concentration levels usually found in real samples.
The method was applied for the determination of NAA in fruits
and the reproducibility of the method was checked in about
100 samples. A quantitative method for the detection of NAA
in fruits that can be complimentary to HPLC methods is provided
in the present paper. These lipid films can be used as portable
sensors for the rapid detection of NAA in fruits by non-skilled
personnel.
[Back to top]
Cholinesterase Biosensor Construction – A Review
M. Pohanka, D. Jun, H. Kalasz and K.
Kuca
Biosensors using cholinesterases as the biorecognition component
have been used to assay organophosphates and carbamates for
a long time. In this review, some strategies convenient for
biosensor construction are presented. Solutions for cholinesterase
immobilization and output signal monitoring are presented
as the basic presumptions for successful biosensor construction.
[Back to top]
Aptabodies – New Type of Artificial Receptors for Detection
Proteins
T. Hianik, A. Porfireva, I. Grman and
G. Evtugyn
We report on a new type of artificial receptor formed by hybridization
of two DNA aptamers for human thrombin (aptabody). This aptasensor
based on multiwalled carbon nanotubes allowed us to detect
thrombin with detection limit of 0.3 nM, which was 3 times
better in comparison with conventional aptamer.
[Back to top]
Determination of Binding Potency of Peptidic Inhibitors of
Grb2 SH2 by Using the Protein-Captured Biosensor Method
F.-D.T. Lung, W.-C. Li, Y.-H. Chang and
H.-M. Chen
The growth factor receptor-binding protein 2–Src homology
2 (Grb2–SH2) domain plays an important role in the oncogenic
Ras signal transduction pathway, therefore, peptidic inhibitors
of the Grb2–SH2 domain has been chosen as our target
for the development of antiproliferative agents. The inhibitory
effects of peptide analogs on the Grb2–SH2 domain have
been determined by using surface plasmon resonance (SPR) technology
developed with the BIACORE biosensor. Recently, we reported
the analysis of interactions between peptides and the GST-Grb2-SH2
that was immobilized on the surface of sensor chip by using
BIACORE biosensor (the protein-immobilized method). Herein,
we analyze interactions of peptides with the GST-Grb2-SH2
that was captured by the anti-GST antibodies immobilized on
the surface of sensor chip (the protein-captured method).
Results obtained by both methods are in good correlation,
indicating the immobilization of GST-Grb2-SH2 on the sensor
chip did not significantly affect the binding of Grb2-SH2
with peptides. Both SPR-based assays are very sensitive bioanalytical
methods and can be applied in screening inhibitors of target
proteins or purifying GST-fusion proteins, however, considering
the efficiency and the cost, the GST-Grb2-SH2-immobilized
method is suggested for routinely determining the binding
potency of inhibitors of Grb2-SH2.
[Back to top]
Circular Dichroism Reveals Sensitivity of Glucagon Solution
Structure to Fluoroalcohols, pH and Ionic Strength
S. Guest, V.V. Ngo and M.A. Hefford
Circular dichroism reports that glucagon in solution becomes
increasingly helical with the addition of fluoroalcohol (which
also decreases solution pH), and with changes in pH and ionic
strength. Given the variability of structure observed, these
data indicate that care must be taken when comparing results
obtained under different solution conditions.
[Back to top]
Changes in Structure and in Interactions of Heat-Treated Bovine
β-Lactoglobulin
S.H.-A. Mousavi, A.-K. Bordbar and
T. Haertlé
Heat stress on structure and ligand binding of β-LG
has been studied by fluorescence, circular dichroism and gel
electrophoresis at pH 6.5. Native PAGE gel electrophoresis
shows that denaturation of β-LG
is reversible up to 75 °C
then it becomes irreversible due to aggregation of β-LG.
Formation of aggregated β-LG
is completed at 95 °C.
Circular dichroism results indicate that formation of aggregated
β-LG
is accompanied by the scrambling of disulfide bonds (creation
of new intramolecular and intermolecular disulfide bridges
and rearrangement of old intramolecular disulfide bridges).
Addition of ethanolic retinol causes a change in polarity
of the solution and favors transformation of the β↔α
structure. In the presence of retinol, the α-helix
content of the secondary structure of heat-treated β-LG
is increased and the major por-tion of its secondary structure
is helical. Fluorescence results show that heat-treated β-LG
at 95 °C
can still bind retinol. The refolding of the tertiary structure
of β-LG
heat-denatured at 95 °C
may recreate a retinol binding site. Surprisingly, the affinity
of the new site for retinol is higher than that of native
β-LG;
however, the apparent molar ratio is lower than one. The binding
properties of β-LG
for terpenoids have been measured after its heat treatment
at 20, 75 and 95 °C.
The intensity of tryptophan emission at 330 nm was changed
only in the case of the interaction with β-ionone.
Other ligands probably cannot bind to β-LG
or they bind in a binding site far from the tryptophan residues,
hence not affecting its fluorescence.
[Back to top]
Urea and Acid-Induced Unfolding of Fatted and Defatted Human
Serum Albumin
P. Salahuddin
Urea induced equilibrium unfolding of fatted and defatted
human serum albumin (HSA) showed that fatty acid stabilizes
native and intermediate states. Similarly acid induced unfolding
of fatted and defatted HSA also showed that fatty acid stabilizes
transitions. These results also showed that five electrostatic
interactions along with one buried carboxyl group of acidic
amino acid are involved in acid induced unfolding of HSA.
[Back to top]
Classification of Amine Type G-Protein Coupled Receptors with
Feature Selection
Q.-B. Gao, C. Wu, X.-Q. Ma, J. Lu and
J. He
G-protein coupled receptors (GPCRs) are involved in various
physiological processes. Therefore, classification of amine
type GPCRs is important for proper understanding of their
functions. Though some effective methods have been developed,
it still remains unknown how many and which features are essential
for this task. Empirical studies show that feature selection
might address this problem and provide us with some biologically
useful knowledge. In this paper, a feature selection technique
is introduced to identify those relevant features of proteins
which are potentially important for the prediction of amine
type GPCRs. The selected features are finally accepted to
characterize proteins in a more compact form. High prediction
accuracy is observed on two data sets with different sequence
similarity by 5-fold cross-validation test. The comparison
with a previous method demonstrates the efficiency and effectiveness
of the proposed method.
[Back to top]
Structural Bioinformatics Study of PNP from Listeria monocytogenes
L.F.S.M. Timmers, R.A. Caceres, L.A. Basso,
D.S. Santos and W.F. De Azevedo, Jr.
This work describes for the first time a model of Purine Nucleoside
Phosphorylase from Listeria monocytogenes (LmPNP).
We modeled the complexes of LmPNP with ligands in
order to determine the structural basis for specificity. Comparative
analysis of the model of LmPNP allowed identification
of structural features responsible for ligand affinities.
[Back to top]
Telmisartan Inhibits Advanced Glycation End Products (AGEs)-
Elicited Endothelial Cell Injury by Suppressing AGE Receptor
(RAGE) Expression Via Peroxisome Proliferator-Activated
Receptor -γActivation
S.-i. Yamagishi, T. Matsui, K. Nakamura,
M. Takeuchi and H. Inoue
Advanced glycation end products (AGEs)-their receptor (RAGE)
axis plays a central role in the pathogenesis of diabetic
microangiopathy. Since the pathophysiological crosstalk between
the AGEs-RAGE system and angiotensin II has also been associated
with diabetic microangiopathy, we examined here whether and
how telmisartan, a unique angiotensin II type 1 receptor blocker
(ARB) with peroxisome proliferator-activated receptor-γ
(PPAR-γ)-modulating
activity, could inhibit the AGEs-elicited endothelial cell
injury by suppressing RAGE expression in vitro. Telmisartan
suppressed RAGE expression at both mRNA and protein levels
in human cultured microvascular endothelial cells (ECs), which
were prevented by GW9662, an inhibitor of PPAR-γ.
Further, telmisartan was found to inhibit up-regulation of
mRNA levels for monocyte chemoattractant protein-1, intercellular
adhesion molecule-1 and vascular endothelial growth factor
in AGEs-exposed ECs. These results suggest that telmisartan
inhibits the AGEs-elicited EC injury by down-regulating RAGE
expression via PPAR-γ
activation. Our present study provides a unique beneficial
aspect of telmisartan. Specifically, it could work as an anti-inflammatory
agent against AGEs via PPAR-γ
activation and may play a protective role against diabetic
microangiopathy.
[Back to top]
Preparation and Characterization of a Novel Recombinant Human
Parathyroid Hormone (1–34) Analog (Gly1-Gln26-rhPTH(1
34)) with Enhanced Biological Activity
X.-C. Xu, S.-D. Zhong, F. Kai, L.-R. Li,
C. Liu, B. Liu and J.-K. Bao
A recombinant human parathyroid hormone (rhPTH) fragment (Gly1-Gln26-rhPTH(1-34))
which contains two amino acids substitutions (Gly1
and Gln26) was acquired through
Escherichia coli expression system using a soluble
fusion protein strategy. The soluble fusion protein MBP-Gly1-Gln26-rhPTH(1-34)
was harvested after purification by Phenyl-Sepharose F.F and
Q-Sepharose F.F chromatographies. Following tobacco etch virus
(TEV) protease cleavage and further purification by SP-Sepharose
F.F chromatography, 30.8 mg/L Gly1-Gln26-rhPTH(1-34)
without tag was obtained with high purity up to 99%. Cyclic
AMP (cAMP) stimulation assay suggested that Gly1-Gln26-rhPTH(1-34)
could increase the biological activity by up to 13.89% and
6.34%. After daily subcutaneous injection (for 13 weeks) of
5, 10 and 20 μg
of Gly1-Gln26-rhPTH(1-34)/1000g
body weight, the mean Bone Material Density (BMD) of ovariectomized
(OVXed) rats increased to 7.95–30.54% and 1.98–23.32%,
compared to control-vehicle group (OVX, P<0.001)
and sham- operated group (SHAM, P<0.01),
respectively.
[Back to top]
Antimicrobial and Anti-Inflammatory Activities of Designed
Antimicrobial Peptide P18 Analogues
Y.H. Nan and S.Y. Shin
To develop antimicrobial peptides having higher bacterial
selectivity than a novel antimicrobial peptide P18, we synthesized
several analogues. The P18 analogues are designed by movement
of the N-terminal Trp2 residue
in P18 (P18-W6, P18-W8
and P18-W15) and the substitution
of the central Pro9 residue
with D-Pro or Nala (P18-Nala9
and P18-D-Pro9). These analogues
retained potent antibacterial activity but displayed less
hemolytic activity than P18. From the viewpoint of their therapeutic
index, P18 analogues had approximate 3- to 7-fold higher bacterial
selectivity compared to P18. The analogues preferentially
bind to bacterial membrane-mimicking negatively charged liposomes
as well as does P18. Their high specificity to negatively
charged phospholipids corresponds well with their high bacterial
selectivity. Furthermore, P18-W6,
P18-W8 and P18-Nala9
induced a significant inhibition in NO production from LPS-stimulated
macrophage RAW264.7 cells, as well as P18. This result suggests
that these peptides appear to have promising therapeutic potential
for future development as a novel anti-inflammatory agent
as well as antimicrobial agent.
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