Development of a peptide substrate for detection of sunn pest damage in wheat flour

BACKGROUND: Since the common protease substrates did not give satisfactory results for the determination of Sunn pestprotease activity in damaged wheat, different peptide substrates derived from the repeated sequences of high molecular weightglutenin subunits were synthesized.RESULTS: Hydrolysis of peptides by pest protease was determined by high-performance liquid chromatography. Among threepeptides having the same consensus motifs, peptide1 (PGQGQQGYYPTSPQQ) showed the best catalytic efficiency. A novel assaywas described for monitoring the enzymatic activity of protease extracted from damaged wheat flour. The selected peptide waslabeled with a fluorophore (EDANS) and quencher (Dabcyl) to display fluorescence resonance energy transfer. The proteolyticactivity was measured by the change in fluorescence intensity that occurred when the protease cleaved the peptide substrate.Furthermore, the assay developed was modified for rapid and easy detection of bug damage in flour. Flour samples weresuspended in water and mixed with fluorescence peptide substrate. After centrifugation, the fluorescence intensities of thesupernatants, which are proportional to the protease content of the flour, were determined.CONCLUSION: The total analysis time for the assay developed is estimated as 15 min. The assay developed permits a significantdecrease in time and labor, offering sensitive detection of Sunn pest damage in wheat flour.WoSPubMe

Enhancing the affinity of SEB-binding peptides by repeating their sequence

WOS: 000311339500007PubMed: 22733526The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12-mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage-display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB-binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12-mer peptide sequences. A 10-fold increase was observed in the binding affinity of one of the two-repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems. (C) 2011 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 145154, 2012.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [108O031]; Hacettepe University Scientific Research FundHacettepe University [09.01.602.003]Contract grant sponsor: Scientific and Technological Research Council of Turkey (TUBITAK); Contract grant number: 108O031; Contract grant sponsor: Hacettepe University Scientific Research Fund; Contract grant number: 09.01.602.00

Glucose determination based on a two component self-assembled monolayer functionalized surfaceenhanced Raman spectroscopy (SERS) probe

Dudak, Fahriye Ceyda/0000-0003-4998-0460; Tamer, Ugur/0000-0001-9989-6123WOS: 000338438500022In this report, we present a new detection method for blood glucose, using gold nanorod SERS, a surface enhanced Raman scattering probe embedded in two component self-assembled monolayers (SAMs). Gold nanorod particles and a gold coated slide surface were modified with the two component SAMs consisting of 3-mercaptophenylboronic acid (3-MBA) and 1-decanethiol (1-DT). The immobilization of 3-MBA/1-DT surface-functionalized gold nanoparticles onto 3-MBA/1-DT modified gold-coated slide surfaces was achieved by the cooperation of hydrophobic forces. Two component SAM functionalized substrates were used as SERS probes, by means of the boronic acid and the alkyl spacer functional groups that serve as the molecular recognition and penetration agents, respectively. The SERS platform surface was characterized by cyclic voltammetry, contact angle measurements, AFM (atomic force microscopy) and Raman spectroscopy. Optimum values of the parameters such as pH, time and (3-MBA/1-DT) molar ratio were also examined for the glucose determination. The analytical performance was evaluated and linear calibration graphs were obtained in the glucose concentration range of 2-16 mM, which is also in the range of the blood glucose levels, and the detection limit was found to be 0.5 mM. As a result, the SERS platform was also used for the determination of glucose in plasma samples.Scientific and Technological Research Council of Turkey (TUBITAK)Turkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [Cost MP 1205-111T983]The authors acknowledge The Scientific and Technological Research Council of Turkey (TUBITAK) with the project number Cost MP 1205-111T983 for funding. The authors would like to thank Prof. Nihal Aydogan and Hande Unsal (Hacettepe University, Department of Chemical Engineering, Turkey) for AFM measurements

The Discovery of Small-Molecule Mimicking Peptides Through Phage Display

Using peptides to achieve the functional and structural mimicry of small-molecules, especially those with biological activity or clear biotechnological applications, has great potential in overcoming difficulties associated with synthesis, or unfavorable physical properties. Combinatorial techniques like phage display can aid in the discovery of these peptides even if their mechanism of mimicry is not rationally obvious.The major focus of this field has been limited to developing biotin and sugar mimetics. However, the full “mimicry” of these peptides has not yet been fully established as some bind to the target with a different mechanism than that of the natural ligand and some do not share all of the natural ligand’s binding partners. In this article, mimicry of small-molecules by phage display-discovered peptides is reviewed and their potential in biochemical and medical applications is analyzed.PubMedWoSScopu