Biotin Site Research Articles

Magneto-controlled fluorescent immunosensor for sensitive determination of biomarker via three-dimensional AuNCs/liposome networks

The three-dimensional liposome networks are fabricated and act as signal amplification modules for the fabrication of a magneto-controlled fluorescent immunosensor, in which antibody-conjugated magnetic bead (MB-Ab), streptavidin-biotin recognition and liposome amplification as the triple signal amplification pathway. The Aptamer-Liposome-AuNCs (Apt-Lip-AuNCs) is employed as a fluorescence signal probe, which can release abundant of AuNCs by Triton-X 100 to produce fluorescence emission signal upon being excited at 369 nm. In the presence of target CEA, the labeled anti-CEA on the MB and the immobilized aptamer on the Apt-Lip-AuNCs sandwich the target CEA. Upon addition of streptavidin and Biotin-Lip-AuNCs, three-dimensional liposome networks are formed by a streptavidin-biotin reaction due to the capture of streptavidin for Biotin-Lip-AuNCs and the residual biotin site of liposome on the Apt-Lip-AuNCs, thereby increasing the loading capacity of AuNCs. CEA can be linearly assayed in the range from 0.05 ng mL−1 to 40 ng mL−1 with a detection limit of 13.2 pg mL−1 (3σ/K). It provided a concise sensing platform for CEA determination and showed a great potential application for on-site testing in early clinical diagnosis.

A new QCM signal enhancement strategy based on streptavidin@metal-organic framework complex for miRNA detection

Sensitive and selective detection of miRNA is of great significance for the early diagnosis of human diseases, especially for cancers. Quartz crystal microbalance (QCM) is an effective tool for detecting biological molecules; however, the application of QCM for miRNA detection is still very limited. One of the great needs for QCM detection is to further improve the QCM signal. Herein, for the first time, we promote a new signal enhancement strategy for the detection of miRNA by QCM. First, a hairpin biotin-modified DNA was used as a probe DNA, which exposes the biotin site when interacting with target miRNA. Then, a streptavidin@metal-organic framework (SA@MOF) complex formed by electrostatic attractions between SA and a MOF was introduced into the QCM detection system. The SA@MOF complexes serve as both a signal amplifier and a specific recognition element via specific biotin-SA interactions. The strategy was applied to the detection of a colorectal cancer marker, miR-221, by using a stable Zr(IV)-MOF, UiO-66-NH2. The detection linear range was 10 fM-1 nM, the detection limit was 6.9 fM, and the relative standard deviation (RSD) (n = 5) was lower than 10% in both simulated conditions and the real serum environment. Furthermore, the detection limit reached 0.79 aM when coupled with the isothermal exponential amplification reaction (EXPAR).

A graphene oxide-based multiplexed fluorescence assay for the detection of protein kinase activity in cell lysates and the evaluation of protein kinase inhibition

We have developed a novel graphene oxide (GO) sensing platform based on enzymatic phosphorylation for the multiplexed detection of protein kinases. The dye-labeled peptide substrate exhibits strong fluorescence in the absence of kinases. When the biotinylated adenosine 5′-triphosphate co-substrate and kinase are present, the peptide substrate is phosphorylated by kinase, and the phosphorylated peptide product carrying both the biotin site and the dye can be assembled onto streptavidin-coated GO via streptavidin-biotin chemistry, leading to fluorescence quenching. Importantly, since GO is a highly efficient quencher for multiple different fluorophores, the platform allows simultaneous detection of multiple protein kinases using different peptide substrates labeled with corresponding dyes. As a proof-of-concept, we demonstrate that this GO sensing platform can simultaneously and selectively detect protein kinase A, protein kinase Abl and protein kinase Src with low detection limits of 0.005U/mL, 0.02U/mL and 0.05U/mL, respectively. Furthermore, the application of this method for detection of multiple protein kinases in biological samples and protein kinase inhibitor screening has also been demonstrated. This approach holds great promise as a routine tool for the high-throughput screening of protein kinases and their inhibitors.

CDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA–protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a ‘ligation site’ for T4 RNA ligase, a ‘biotin site’ for solid-phase handling, a ‘reverse transcription primer site’ for the efficient and rapid conversion from an unstable mRNA–protein fusion (mRNA display) to a stable mRNA/cDNA–protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a ‘restriction enzyme site’ for the release of a complex from the solid support. This enables not only stabilizing mRNA–protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Controlling Binding Site Densities on Glass Surfaces

The density of surface-immobilized ligands or binding sites is an important issue for the development of sensors, array- or chip-based assays, and single-molecule detection methods. The goal of this research is to control the binding site density of reactive ligands on surfaces by diluting surface amine groups in self-assembled and cross-linked monolayers on glass prepared from solutions containing very low concentrations of (3-aminopropyl)triethoxysilane (APTES) and much higher concentrations of (2-cyanoethyl)triethoxysilane. The surface amine sites are suitable for attaching labels and ligands by reaction with succinimidyl ester reagents. Labeling the amine sites with fluorescent molecules and imaging the single molecules with fluorescence microscopy provides a means of determining the density of amine sites on the surface, which were incorporated into the self-assembled monolayer with micrometer spacings in proportion to the concentration of APTES in the synthesis. Biotin ligands were also bound to these surface amine sites using a succinimidyl ester linker, and the immobilized biotin was then reacted with either streptavidin-conjugated gold colloid particles or fluorescently labeled neutravidin. Imaging of these samples yields consistent amine and biotin site coverages, indicating that quantitative control and chemical conversion of binding sites can be achieved at very low (<10(-7)) fractions of a monolayer.

Preparation and characterization of active site protected poly(ethylene glycol)–avidin bioconjugates

Avidin was modified with poly(ethylene glycol) in the presence of a biotin binding site protective agent synthesised by imminobiotin conjugation to branched 20 kDa PEG. Avidin was incubated with imminobiotin-PEG and reacted with high amounts of 5, 10 or 20 kDa PEG to modify the protein amino groups. Circular dichroism demonstrated that the extensive PEGylation does not alter the protein conformational structure. The affinity of avidin-PEG conjugates for biotin and biotinylated antibodies depended on the PEG size or the use of a protective agent. Avidin-PEG 10 and 20 kDa prepared in the presence of imminobiotin-PEG maintained 100% of the native affinity for biotin. The 5 kDa PEG derivative and the ones obtained without biotin site protection maintained 79-85% of the native affinity. The affinity for biotinylated antibodies decreased to 35% when the conjugation was performed without imminobiotin-PEG, while the conjugates obtained with high-molecular-weight PEGs in the presence of protective agent displayed high residual affinity. All conjugates possessed negligible antigenicity and immunogenicity. PEGylation greatly prolonged the avidin permanence in the circulation, reduced its disposition in the liver and kidneys and promoted accumulation into solid tumors. PEGylation was found to prevent the protein cell uptake, either by phagocytosis or pinocytosis.

Voltammetric detection of avidin and biotin by biotin labeled with cysteine

An avidin-biotin assay was developed from a voltammetric procedure using biotin labeled with cysteine. Mercury(II) as a marker was used to detect avidin and biotin, because the oxidation wave of mercury decreases when the cysteine part of labeled biotin(LB) complexes with mercury(II).The formation of the mercury(II)-cysteine complex is suppressed when the LB binds to the biotin site of avidin. Accordingly, the concentration of avidin can be estimated from the increasing mercury peak current. Detection of biotin is also carried out by a competitive reaction of biotin and the LB to the binding site on avidin, where the addition of biotin decreases the peak current of mercury. Limits of detection for avidin and biotin were in the 10–9 mol/L range. The length of the spacer between the cysteine and biotin was investigated. It was observed that the strength of binding increased with increasing length of spacer. Size considerations rules out steric influences, so it is suggested that the binding constant depends on hydrophobic interactions in the binding site.

Biotin binding changes the conformation and decreases tryptophan accessibility of streptavidin

Biotin binding reduces the tryptophan fluorescence emissions of streptavidin by 39%, blue shifts the emission peak from 333 to 329 nm, and reduces the bandwidth at half height from 53 to 46 nm. The biotin-induced emission difference spectrum resembles that of a moderately polar tryptophan. Streptavidin fluorescence can be described by two lifetime classes: 2.6 nsec (34%) and 1.3 nsec (66%). With biotin bound, lifetimes are 1.3 nsec (26%) and 0.8 nsec (74%). Biotin binding reduces the average fluorescence lifetime from 1.54 to 0.88 nsec. Biotin does not quench the fluorescence of indoles. The fluorescence changes are consistent with biotin binding causing a conformational change which moves tryptophans into proximity to portions of streptavidin which reduce the quantum yield and lifetimes. Fluorescence quenching by acrylamide revealed two classes of fluorophores. Analysis indicated a shielded component comprising 20-28% of the initial fluorescence with (KSV + V) less than or equal to 0.55 M-1. The more accessible component has a predominance of static quenching. Measurements of fluorescence lifetimes at different acrylamide concentrations confirmed the strong static quenching. Since static quenching could be due to acrylamide binding to streptavidin, a dye displacement assay for acrylamide binding was constructed. Acrylamide does bind to streptavidin (Ka = 5 M-1), and probably binds within the biotin-binding site. In the absence of biotin, none of streptavidin's fluorescence is particularly accessible to iodide. In the presence of biotin, iodide neither quenches fluorescence nor alters emission spectra, and acrylamide access is dramatically reduced. We propose that the three tryptophans which always line the biotin site are sufficiently close to the surface of the binding site to be quenched by bound acrylamide. These tryptophans are shielded from iodide, most probably due to steric or ionic hindrances against diffusion into the binding site. Most of the shielding conferred by biotin binding can be attributed to the direct shielding of these residues and of a fourth tryptophan which moves into the binding site when biotin binds, as shown by X-ray studies (Weber et al., 1989).

A rapid purification method for rat liver pyruvate carboxylase and amino acid sequence analyses of NH 2-terminal and biotin peptide

A rapid method for the purification of pyruvate carboxylase from rat liver has been developed. The method involves extraction of the enzyme from frozen liver powder followed by polyethylene glycol fractionation and avidin-affinity chromatography. The purified enzyme has a specific activity of 9–10 μmol/min/mg protein when assayed at 22 °C in the presence of acetyl-CoA. Polyacrylamide gel electrophoresis of the preparation in the presence of sodium dodecyl sulfate showed the presence of one protein band with an estimated M r 125,000 and no significant contamination by other biotin-containing enzymes. In addition to being rapid, the method is advantageous because prior isolation of mitochondria is not necessary. Using these preparations we have determined the sequence of the first 15 amino acids from the NH 2-terminal end of the molecule to be SerGlyProValAlaPro LeuAsnValLeuLeuLeuGluTyrPro. The sequence of the 24 amino acid residues around the biotin site was determined to be GlyAlaProLeuValLeuSerAlaMet -biocytinMetGluThr ValValThrSerProThrGluGlyThrIleArg.

Sequence homology around the biotin-binding site of human propionyl-CoA carboxylase and pyruvate carboxylase

Biotin-dependent carboxylases require covalently bound biotin for enzymatic activity. The biotin is attached through a lysine residue, which in a number of bacterial, avian, and mammalian carboxylases, is found within the conserved sequence AlaMetLysMet. We have determined the partial nucleotide sequence of cDNA clones for human propionyl-CoA carboxylase and pyruvate carboxylase. The predicted amino acid sequence of both these proteins contains the conserved tetrapeptide 35 residues from the carboxy terminus. In addition, both proteins contain the tripeptide, ProMetPro, 26 residues toward the amino terminus from the biotin attachment site. The overall amino acid homology through this region is 43%. Similar findings have been made for the biotin-containing polypeptides of transcarboxylase of Propionibacterium shermanii and acetyl-CoA carboxylase of Escherichia coli ( W. L. Maloy, B. U. Bowien, G. K. Zwolinski, K. G. Kumar, and H. G. Wood (1979) J. Biol Chem. 254, 11615–11622). The implications of this sequence conservation with regard to the function and evolution of biotin-dependent carboxylases is discussed. We propose that the 60 amino acids surrounding the biotin site are bounded by a proline “hinge” and the carboxy terminus has remained conserved as a result of constraints imposed by biotinylation of the enzyme.

Molecular cloning of a cDNA for human pyruvate carboxylase. Structural relationship to other biotin-containing carboxylases and regulation of mRNA content in differentiating preadipocytes.

An oligonucleotide probe specific for the amino acid sequence at the biotin site in pyruvate carboxylase was used to screen a human liver cDNA library. Nine cDNA clones were isolated and three proved to be pyruvate carboxylase clones based on nucleotide sequencing and Northern blotting. The biotin site amino acid sequence of human pyruvate carboxylase agreed perfectly with that of the sheep enzyme in 14 consecutive positions. The highly conserved amino acid sequence, Ala-Met-Lys-Met, found at the biotin site in most biotin-containing carboxylases was also present in human pyruvate carboxylase. The termination codon was located 35 residues 3' to the lysine residue at which the biotin is attached. Therefore, the biotin cofactor is covalently linked near the carboxyl-terminal end of the carboxylase protein. These data are consistent with that observed for other biotin-containing carboxylases and strongly suggests that the genes encoding the biotin-containing carboxylases may have evolved from a common ancestral gene. Northern blotting of mRNA isolated from human, baboon, and rat liver demonstrated that the pyruvate carboxylase mRNA was 4.2 kilobase pairs in length in all species examined. Southern blot analysis of genomic DNA isolated from human-Chinese hamster somatic cell hybrids localized the pyruvate carboxylase gene on the long arm of human chromosome 11. The human cDNA was also used to quantitate pyruvate carboxylase mRNA levels in a differentiating mouse preadipocyte cell line. These data demonstrated that pyruvate carboxylase mRNA content increased 23-fold in 7 days after the onset of differentiation.

Formation of Nε-(biotinyl)lysine in biotin enzymes

The role of biotin in carboxyl transfer reactions has been the subject of considerable investigation for a number of years and the mechanisms of the enzymes that catalyze these reactions are known in some detail. The biotin is covalently attached to the enzyme through an amide bond that links the carboxyl group of the valeric acid side chain of biotin to the ɛ-amino group of a lysine residue in the enzyme, yielding a biocytin residue. Although the mechanisms of catalysis of biotin-dependent enzymes have much in common, their structures show considerable diversity. The enzymes vary from structures having the donor site, acceptor site, and biotin attachment site on a single polypeptide chain, as in pyruvate carboxylase of animals, to ones that have each of these three functional sites on separate polypeptides, as in transcarboxylase of Propionibacterium shermanii or acetyl-CoA carboxylase of Escherichia coli . The most common structural form for biotin enzymes appears to be between these two extremes, with the biotin and carboxylation site on one polypeptide and the acceptor site apparently located on a second polypeptide. The covalent attachment of biotin to a specific lysine(s) in apoenzymes is catalyzed by holoenzyme synthetase. The chapter discusses the purification and properties of holocarboxylase synthetase. There are two general methods that have been used to determine the activity of holocarboxylase synthetase. The first procedure follows the incorporation of radioactive biotin into the appropriate apoenzyme directly and the second procedure monitors the appearance of the resultant active holoenzyme.

The carboxyl transferase component of acetyl CoA carboxylase: structural evidence for intersubunit translocation of the biotin prosthetic group.

An essential protein component of acetyl CoA carboxylase, isolated and extensively purified from cell-free extracts of Escherichia coli, has been identified as malonyl CoA: d-biotin carboxyl transferase. This enzyme, which does not contain covalently-bound biotin, catalyzes carboxyl transfer from malonyl CoA to free d-biotin, a model reaction for the second step in the carboxylation of acetyl CoA. The transcarboxylation product, after stabilization by methylation, was identified as 1'-N-carboxy-d-biotin dimethyl ester. These results indicate the presence of a biotin site on the carboxyl transferase, distinct from that on the biotin carboxylase, which carries out the first step in the overall process. In addition, the carboxyl transferase catalyzes a slower abortive decarboxylation of malonyl CoA, thus indicating that carboxyl abstraction and protonation do not require the participation of biotin. It is now evident that the half-reactions of acetyl CoA carboxylation are catalyzed by biotin carboxylase and carboxyl transferase. Both components are devoid of biotin and have specific binding sites for free d-biotin, as well as for their respective substrates; hence, the acetyl CoA carboxylation mechanism must involve intersubunit translocation of the carboxylated biotinyl group, which is bound covalently to carboxyl-carrier-protein, a noncatalytic polypeptide.