Isotope‐Coded Affinity Tagging Technique Using Avidin Functional Affinity Electrophoresis: An Alternative to an Avidin Affinity Column

Avidin functional affinity electrophoresis (AFAEP) is substituted for an avidin affinity column (AAC) to capture biotinylated peptides in the Isotope‐Coded Affinity Tagging (ICAT) technique which is a valuable tool in quantitative proteomics. In this new technique, the AFAEP‐captured ICAT‐labeled biotinylated peptides are extracted with the biotin tag intact from the polyacrylamide gel piece with aqueous 95% formamide (pH 8.2) at 65 °C for 20 min, and then detected by a matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometer. Bovine serum albumin (BSA) and the ¹²C‐ and ¹³C‐ICAT reagents are used to test this AFAEP‐ICAT technique. The results show that both AFAEP and AAC methods provide quantitative information of the relative amounts of ¹²C‐ and ¹³C‐ICAT‐labeled biotinylated tryptic peptides of BSA in a sample. Compared with AAC, the AFAEP is cheaper to perform, more stringent in capturing the biotinylated peptides, and capable of simultaneously processing multiple samples.     

Immobilization of peroxidase on SiO2 surfaces with the help of a dendronized polymer and the avidin-biotin system

Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications.     

Functionalization of Silk Fibroin with NeutrAvidin and Biotin

New methods are needed to modify silk biomaterials with bioactive molecules for tissue engineering and drug delivery. In the present study, silk fibroin in solution or in microsphere format was coupled with NeutrAvidin via carbodiimide chemistry. Silk fibroin retained its self‐assembly features after reaction. It was found that more than four NeutrAvidin molecules bound to one silk molecule. Non‐specific binding of biotin or NeutrAvidin to silk microspheres could be reduced by pre‐treatment of the microspheres with BSA or post‐treatment with detergent. The NeutrAvidin‐coupled silk microspheres were coupled with biotinylated anti‐CD3 antibody and the functionalized microspheres were able to specifically bind to the CD3 positive T‐lymphocytic cell line Jurkat.

     

抗生物素蛋白与DNA相互作用的单分子研究

抗生物素蛋白(avidin)在生物单分子实验中被广泛用于DNA与修饰表面的连接,同时avidin也可作为一种DNA载体用于基因治疗中.本文利用原子力显微镜(AFM)、动态光散射(DLS)、单分子磁镊(MT)技术系统地研究了avidin与DNA之间的相互作用,以及avidin引起DNA凝聚的机理.首先通过AFM对avidin-DNA复合体形貌进行观察,发现不但有avidin导致DNA凝聚的环状形貌,同时也存在avidin自身聚集引起的DNA凝聚现象,通过定量分析,发现其凝聚尺寸越来越小,而当avidin浓度大于2 ng·μL~(-1)时,其凝聚尺寸又突然变大.DLS实验结果也显示了同样的规律,伴随着avidin浓度的升高,DNA的粒径大小从大约170 nm减小到125 nm左右,其电泳迁移率由-2.76(10~(-4)cm~2·V~(-1)·s~(-1))变化到-0.1(10~(-4)cm~2·V~(-1)·~(-1)).此外,通过MT技术的力谱曲线变化,发现avidin导致的DNA凝聚与其他多价离子相比,长度的变化曲线几乎呈线性变化,偶尔存在少而小的阶跃,这种变化趋势与组蛋白的变化曲线更相似.因此可以判断,avidin导致DNA凝聚是由avidin与DNA的静电吸引和avidin自身聚集两种相互作用引起的.     

A novel,simple and sensitive ligand affinity capture method for detecting molecular interactions by MALDI mass spectrometry

A simple and sensitive ligand affinity capture method (LAC) was developed to detect biotinylated biomolecules bound to a biotin–avidin base by matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI ToF MS). Glass slides covered with a metal film for MALDI MS applications were treated with amino‐silane and derivatized with biotin followed by binding of avidin. Washing buffers with high ionic strength increased the specificity of the subsequent binding of biotinylated biomolecules to the avidin layer. A combined thin layer‐dried droplet method using α‐cyano‐4‐hydroxycinnamic acid (CHCA) in acetone or ethyl acetate resulted in the most intense ions of biotinylated polymyxin B, whereas the matrix conditions did not influence the detection of angiotensin II. Addition of biotinylated biomolecules in the low femtomole to low picomole range resulted in sufficient ion intensity for detection by the LAC method. The LAC concept was extended by binding of biotinylated lipopolysaccharide to the biotin–avidin base followed by preferential capture and specific detection of the binding antagonist polymyxin B. Copyright © 2008 John Wiley & Sons, Ltd.     

Biotinyl-Glucose-6-Phosphate Dehydrogenase Preparation,Kinetics, and Modulation by Avidin

The kinetics of free glucose-6-phosphate dehydrogenase (G-6-PDH), biotinylated G-6-PDH, and biotinylated G-6-PDH complexed with avidin were investigated. The kinetics of the free enzyme were consistent with a sequential rather than a ping-pong mechanism. The kinetics of the biotinylated enzyme were similar to that of the free enzyme, but the kinetic constants were different; theK _m value for NADP was halved, whereas theK _m for G-6-P decreased only slightly. In the presence of avidin, theK _m of biotinylated G-6-PDH for G-6-P nearly doubled whereas theK _m for NADP did not change significantly. Avidin complexed with biotinylated G-6-PDH inhibited the enzyme from acting. Based upon these reactions, it was possible to devise assays for either free biotin or free avidin using biotinylated G-6-PDH as the indicator enzyme. Concentrations of biotin between 40 and 60 mg/mL, or of 25–95 Μg/mL of avidin could be measured within 2 min through the use of biotinylated G-6-PDH.     

Effects of an avidin-biotin binding system on chondrocyte adhesion and growth on biodegradable polymers

Cell adhesion to a scaffold is a prerequisite for tissue engineering. Many studies have been focused on enhancing cell adhesion to synthetic materials that are used for scaffold fabrication. In this study, we applied an avidin-biotin binding system to enhance chondrocyte adhesion to biodegradable polymers. Biotin molecules were conjugated to the cell membrane of chondrocytes, and mediated cell adhesion to avidin-coated surfaces. We demonstrated that immobilization of biotin molecules to chondrocyte surfaces enhanced cell adhesion to avidin-coated biodegradable polymers such as poly(L-lactic acid), poly(D,L-lactic acid), and polycaprolactone, compared to the adhesion of normal chondrocytes to the same type of biodegradable polymer. The biotinylated chondrocytes still maintained their proliferation ability. This study showed the promise of applying the avidin-biotin system in cartilage tissue engineering. [diagram in text].     

Biotin functionalized poly(sulfonic acid)s for bioconjugation: In situ binding monitoring by QCM‐D

We describe the synthesis of biotin end functionalized poly(sulfonic acid)s via living radical polymerization (LRP) for conjugation to Avidin. Quartz crystal microbalance (QCM‐D) and competitive binding studies were used to confirm this conjugation. A biotin initiator for copper‐mediated LRP was used to provide acrylamide and methacrylate based polymers with the functional end group. This investigation revealed that 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid was not a suitable monomer in its acid form but was successfully used in its sodium salt form. A second monomer, 3‐sulfopropylmethacrylate as the potassium salt was also studied and both monomers produced polymers with polydispersities <1.3 and 1.4, respectively. Evolution of molecular weight with respect to time indicated that the polymerization of the acrylamide polymer is controlled. Quartz crystal microbalance with dissipation monitoring was used to confirm that the biotinylated polymers were able to bind to Avidin in situ. The gold surface of a quartz crystal was chemically modified resulting in a stable monolayer of Avidin; the biotinylated polymers were passed over the functionalized surface and their grafting ability was examined. A competitive binding evaluation was undertaken with 2‐(4‐hydroxyphenylazo)benzoic acid (HABA) dye to provide visual verification of conjugation. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Accessing the applicability of polarized protein‐specific charge in linear interaction energy analysis

The reliability of the linear interaction energy (LIE) depends on the atomic charge model used to delineate the Coulomb interaction between the ligand and its environment. In this work, the polarized protein‐specific charge (PPC) implementing a recently proposed fitting scheme has been examined in the LIE calculations of the binding affinities for avidin and β‐secretase binding complexes. This charge fitting scheme, termed delta restrained electrostatic potential, bypasses the prevalent numerical difficulty of rank deficiency in electrostatic‐potential‐based charge fitting methods via a dual‐step fitting strategy. A remarkable consistency between the predicted binding affinities and the experimental measurement has been observed. This work serves as a direct evidence of PPC's applicability in rational drug design. © 2014 Wiley Periodicals, Inc.     

Preparation of an Organized Film Composed of Polymers,Avidin, and Concanavalin A and Its Binding Properties

The multilayer thin films composed of polymers, avidin, and concanavalin A (Con A) were prepared on the surface of a quartz slide by alternate deposition of anionic and cationic polymers and the proteins. The loading of avidin and Con A in the film was evaluated spectroscopically by using Texas Red tagged avidin and Con A. Avidin molecules assembled in the film caused its binding activity toward biotin and analogue 2-(4′-hydroxyphenylazo)benzoic acid to be retained. The polymer layers contributed to improving stability of the protein film.

Polymer/Con A/avidin composite film on a solid surface.