High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.     

Post-biotinylation of photocrosslinking by Staudinger-Bertozzi ligation of preinstalled alkylazide tag

Post-biotinylation of the alkyl azide derivative of trifluoromethyl phenyldiazirine (TPD) was elucidated to apply a photoaffinity biotinylation technique. A photo-modified polyvinilidene difluoride (PVDF) membrane was used as a photolabeled component and we introduced biotin by Staudinger-Bertozzi ligation. The 15 pmol amount of biotinylated reagent was still effective for the visualization of cross-linked product on the matrix. The results show the potential utility of alkyl azide carrying TPD derivatives in the application of photoaffinity biotinylation, which could be useful for the ligands with tight structural requirements.     

Biotinylation of Peptides/Proteins Using Biocytin Hydrazide

Biocytin hydrazide is widely used to biotinylate the carbohydrate moieties of glycoproteins. In this study, however, biocytin hydrazide was found to be able to directly biotinylate peptides and proteins. This phenomenon may cause false identification of non‐glycopeptides/non‐glycoproteins as glycopeptides/glycoproteins. Here, we report a systematic investigation of the reaction of peptides/proteins with biocytin hydrazide. Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry is used to analyze the biotinylation reaction between peptides/proteins and biocytin hydrazide. Peptides/proteins were reacted with biocytin hydrazide in diverse solvent systems with different biocytin hydrazide concentrations for up to 96 h at temperatures ranging from 4 °C to 65 °C. Singly biotinylated or multiply biotinylated peptides/proteins are observed. The efficiency of the biotinylation reaction increases with higher temperature, higher biocytin hydrazide concentration, or longer reaction time. The influence of buffer pH on the biotinylation reaction of peptides/proteins is less pronounced. The biotinylation efficiency is optimum at neutral pH. Data suggests that the peptides are biotinylated as efficiently as proteins. The observation that peptides/proteins condense only with biocytin hydrazide, 2‐iminobiotin hydrazide, adipic dihydrazide and phenyl hydrazine but not with biocytin HCl and 2‐iminobiotin, indicates that the biotinylation reaction of peptides/proteins occurs with the hydrazide moiety but not with biotin moiety of the biotinylated reagent. The postsource decay data of biotinylated P₁₄R indicates that biocytin hydrazide condenses with the guanidino group of arginine's side chain of P₁₄R, indicating that besides N‐terminal and lysine residue of peptides/proteins, arginine residue is capable of reacting with biocytin hydrazide.     

Development of a solid-supported biotinylation reagent for efficient biotin labeling of SH groups on small molecules

We report here the design and synthesis of a novel and selective SH-group biotinylating reagent, KSH-1 (1), for the biotinylation of small molecules using solid phase chemistry. The results demonstrate that 1 efficiently biotinylated a small molecule, captopril, and afforded the product in high yield and purity.     

Protein purification using combined streptavidin (or avidin)-Sepharose and thiopropyl-Sepharose affinity chromatography.

The major problem usually encountered in the application of the (strept)avidin-biotin system to the purification of proteins (or other biological molecules) lies in the difficult reversion of the interaction between immobilized (strept)avidin and the adsorbed biotinylated protein. Among the proposed solutions is the selective biotinylation of the entity to be purified by a disulphide-containing biotinylated reagent which allows its recovery from (strept)avidin gels by dithiothreitol (DTT) treatment. As emphasized by the example of angiotensin II receptor purification, achieved using this strategy, optimum reduction of this disulphide bridge may require improvement of its accessibility using denaturating agents such as sodium dodecyl sulphate or urea. However, these agents release important amounts of (strept)avidin. Two general ways of solving this problem are proposed. One solution takes advantage of the absence of cysteine in the streptavidin sequence: the protein to be purified is selectively readsorbed to thiopropyl-Sepharose through the thiol function generated on DTT cleavage of the biotinylated reagent. The other solution is an empirical approach to make possible the use of avidin, which possesses cysteine residues: combined avidin-Sepharose and thiopropyl-Sepharose chromatography proved efficient when carried out in the presence of urea as denaturing agent.     

Separation and direct detection of raw and gelatinized starch hydrolyzing activities of glucoamylase on isoelectric focusing gels

A procedure to detect raw and gelatinized starch activities of glucoamylase on isoelectric focusing (IEF) gels by using 2, 3, 5-triphenyltetrazolium chloride is described. The reagent reacts with the reducing group of glucose released by glucoamylase from the substrate starch. Using the reaction, production of glucoamylase by Aspergillus niger was detected on 10% IEF gels within a pH range of 2.5-9.5. Since the method can detect raw and gelatinized starch activities of glucomylase associated with 1 microg protein, it will be useful for enzyme engineering studies that involve screening of various mutations.     

A native, affinity-based protein blot for the analysis of streptavidin heterogeneity: consequences for the specificity of streptavidin mediated binding assays

Commercial preparations of streptavidin, a bacterial biotin-binding protein, were analyzed by isoelectric focusing combined with an affinity-based protein blot using biotinylated, protein-saturated nitrocellulose. The colorimetrical detection of streptavidin with biotinylated alkaline phosphatase allows the selective visualization of streptavidin molecules with at least two active biotin-binding sites. Dependent on the preparation, seven to sixteen streptavidin forms were found with isoelectric points ranging from 5 to 8. Molecular weight analysis of the subunits of streptavidin showed that the observed heterogeneity was mainly due to limited proteolysis, which does not destroy the biotin-binding activity. The preparations differed also in the nonspecific reactivity of streptavidin with single-stranded DNA, bovine serum albumin and Tween 20. No relationship was observed between heterogeneity and non-specific binding activity. Data obtained from protein blots onto nitrocellulose saturated with single-stranded DNA showed that it cannot be excluded that streptavidin with only a single active biotin-binding site is mainly responsible for the nonspecific reactivity of some streptavidin preparations.     

Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis

A faster and more convenient method is required for the detection of recombinant erythropoietin (Epo) in human body fluids. In the present study we wanted to elucidate the principal suitability of immunoaffinity capillary electrophoresis (CE) in this respect. CE offers itself as a high-speed, high-throughput technique provided a suitable affinity reagent is available. We chose monoclonal antibody 5F12 from Amgen which binds to a conformation-independent epitope in the N-terminal region of the human Epo protein. For CE with laser-induced fluorescence detection it was necessary to produce fluorescently labelled antibody with one single antigen binding site. Monomeric antigen-binding fragments (Fab) were obtained by site-selective cleavage of the pure antibody and labelled with the fluorescent dye, Alexa Fluor 488. The mixture of labelled isomers was partially resolved by ion exchange HPLC and isoelectric focusing. The fluorescent Fab could be used to detect erythropoietin by immunoaffinity capillary isoelectric focusing and zone capillary electrophoresis via its antigen complex.Abbreviations BGE background electrolyte - CE capillary electrophoresis - Epo Erythropoietin - Fab antigen-binding fragment - FITC fluorescein isothiocyanate - IEF isoelectric focusing - mAb monoclonal antibody - PBS phosphate-buffered saline - rHuEpo recombinant human erythropoietin - scFv (recombinant) single chain variable fragment - SDS-PAGE denaturing polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate - ECL enzyme-coupled chemoluminescence - v_H variable domain - c_H1–3 constant domains of an antibody's heavy chain     

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.     

Isoelectric focusing in a drop

A novel approach to molecular separations is investigated using a technique termed droplet-based isoelectric focusing. Drops are manipulated discretely on a superhydrophobic surface, subjected to low voltages for isoelectric focusing, and split-resulting in a preparative separation. A universal indicator dye demonstrates the generation of stable, reversible pH gradients (3-10) in ampholyte buffers, and these gradients lead to protein focusing within the drop length. Focusing was visually characterized, spectroscopically verified, and assessed quantitatively by noninvasive light scattering measurements. It was found to correlate with a quantitative model based on 1D steady-state theory. This work illustrates that molecular separations can be deployed within a single open drop, and the differential fractions can be separated into new discrete liquid elements.     

Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel

Rat liver glutathione S-transferases were partially purified using S-hexyl glutathione affinity chromatography, followed by native isoelectric focusing employing a pH 7-11 or pH 3-10 gradient. Proteins were excised and eluted from the gel for determination of subunit composition using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In separate experiments, isoelectric focusing gels were equilibrated with a sodium dodecyl sulfate-containing buffer at high pH, and proteins on the gel were electroblotted onto a polyvinylidene difluoride membrane, utilizing graphite plates as electrodes. The membrane-bound proteins were visualized by Coomassie Brilliant Blue staining. The protein bands were then excised from the membrane and inserted into a gas phase sequenator for direct sequencing. N-Terminal sequences thus determined were compared with published cDNA sequences. The isoelectric points (pIs) and positions on the isoelectric focusing gel of Yb1Yb1, Yb1Yb2 and Yb2Yb2 subunits were determined. We have also located on the pH 3-10 focusing gel an N-terminal blocked glutathione S-transferase which has a molecular weight similar to Yb subunits.     

Haptoglobin subtyping by isoelectric focusing in miniaturized polyacrylamide gels rehydrated in presence of 2-mercaptoethanol

A fast isoelectric focusing method for routine haptoglobin (Hp) subtyping is presented. This method is based on isoelectric focusing, under reducting conditions, of neuraminidase-treated plasma samples by using dry miniaturized (interelectrode distance: 55 mm) polyacrylamide gel, rehydrated in presence of 2-mercaptoethanol and a mixture of pharmalyte carrier ampholytes (pH 4-6.5 and pH 6-8) followed by immunoblotting. The presence of 2-mercaptoethanol in the gel prevented refolding of the Hp alpha and Hp beta chains during focusing, making it possible to obtain a sharp Hp band pattern with a clear separation of the different Hp alpha allelic products (1S, 1F, 2FS, 2SS and 2FF). A population study carried out with 250 unrelated individuals living in Central Spain is also presented.     

Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection

Agarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy-to-handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti-human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.     

Electrochemical characterization of screen-printed carbonaceous electrodes for the determination of peroxidase activity in novel screen-printed flow-through modules

A novel totally screen-printed flow-through cell for immunoanalysis is presented. It contained screen-printed carbonaceous electrodes, which allowed the determination of peroxidase activity through the electrochemical reduction of p-benzoquinone. As different electrode materials differ strongly in their electrochemical properties, electrodes resulting from various screen-printable carbonaceous pastes were characterized using the hydroquinone/ p-benzoquinone redox couple. For most of the electrodes, cyclic voltammogram peak separations of between 550 and 670 mV were observed indicating only quasi-reversible electrochemical behavior. This was confirmed by variation of the peak separation with scan rate. Heterogeneous electron transfer rates of ca. 0.5 - 1 x 10(-3) cm s(-1) and electrochemical activation energies of ca. 20 kJ mol(-1) were found. These flow-through cells were not only applied to electrochemical peroxidase activity determinations but also, in combination with a separate detector, as affinity reactors. After biotinylation of screen-printed layers, streptavidin and then biotinylated peroxidase could be bound. However, as signals were only 10-20% of those obtained with a column filled with biotinylated glass beads, only the screen-printed electrochemical detector was applied to the detection of antibodies against the African Swine Fever Virus.     

Electrophoretic study of conformational changes of a human soluble beta-D-galactoside-binding lectin upon storage.

Human brain lectin (HBL), a beta-galactoside specific soluble lectin, was purified by affinity chromatography. An alkylated derivative of this lectin was also prepared. Both native and modified molecules were conserved at -20 degrees C in the presence or absence of beta-mercaptoethanol, a reducing agent which was described to maintain the lectin activity in vitro or in the presence of beta-mercaptoethanol and lactose. The impact of storage conditions, over one year, on the native and derivated lectins, was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing and titration curve, using the PhastSystem (Pharmacia). Western-blot analysis using an anti-HBL antibody and size-exclusion high performance liquid chromatography were used to complete the study. The subunit M(r)s were estimated before freezing (T0) and after three and twelve months (T3, T12). They were comparable for all preparations. In all samples tested, isoelectric focusing demonstrated the existence of at least three acidic proteins, with the pI ranging between 4.7-4.9. Titration curves clearly showed pH-dependent conformational changes, resulting in a panel of differently charged molecular species, some of which may be related to different oxidative states of the cysteine residues. We concluded that lectin can be stored at -20 degrees C for at least one year before use as a reagent since the modifications revealed by electrophoretic analysis do not alter the hemagglutination activity and carbohydrate binding properties. The immunoreactivity also remained unchanged.     

Synthesis of biotinylated diazinon: Lessons learned for biotinylation of thiophosphate esters

Biotinylation permits recovery of a molecule from a complex mixture, with commercially available streptavidin containing products (such as streptavidin-coated beads). As part of a larger effort to evaluate reagents capable of degrading diazinon, a thiophosphate insecticide, we pursued biotinylation of this molecule. Our strategy focused on replacing a single thiophosphate ethyl ester with an ester linkage that contains biotin. Multiple approaches—using published methods—were unsuccessful and resulted in no reactivity, or degradation of starting material. Here, we report a successful strategy for the synthesis of biotinylated diazinon, which is likely applicable to alternative thiophosphate esters and other biotinylated molecules.     

A method for recovery of native, clonally-restricted immunoglobulins from agarose gels

Multiple low level, clonally-restricted, immunoglobulins (Ig) are commonly encountered on routine serum protein electrophoresis by clinical laboratories using high resolution zone electrophoresis on agarose. We sought a method for recovering the clonally-restricted Ig, in native configuration, from clinical laboratory gels as a first step in the investigation of its clinical significance. We found that a two-stage electrophoretic procedure gave consistently good recoveries. After routine agarose gel electrophoresis, portions of the electropherogram, containing clonally-restricted Ig, were excised and subjected to flatbed isoelectric focusing in agarose to enhance separation of the individual antibody clonotypes. Multiple slabs, containing the same clonally-restricted Ig, could be cut from adjacent tracks (i.e., tracks loaded with the same specimen) on the zone electropherogram and applied to a single track on the focusing gel to improve separation and increase yields. The focused gels were cut to isolate slabs containing individual clonotypes. These slabs were washed to remove carrier ampholytes and held at -20 degrees C overnight. Ig was extracted from the thawed gels, with 61-68% recovery, by ultracentrifugation following physical disruption of the gel. Antigen binding activity of the recovered Ig was verified by rate nephelometry. Clonally-restricted antibodies were successfully isolated from an immune animal serum by this procedure and biotinylated for use as probes on Western blots.     

Dynamics of gel isoelectric focusing with ampholytic dyes monitored by camera in real-time

The dynamics of gel isoelectric focusing were studied by using amphoteric low-molecular-mass colored substances (isoelectric point markers). The polyacrylamide gel in slab format was in direct contact with the electrodes. In addition to isoelectric focusing with a pH gradient composed of synthetic carrier ampholytes, pH gradients created by simple buffers of acetic acid, 2-(N-morpholino)ethanesulfonic acid, histidine and N,N,N',N'-tetramethylethylenediamine were applied. The progress of the electrofocusing process was monitored by a charge-coupled device camera and video recording. The gradient profile and dynamics were approximated from the positions of isoelectric point markers, which were focused both on boundaries between individual zones of simple buffers and within the zones themselves. The obtained animated records enabled the observation of the entire real focusing run within fractions of a minute, which is useful both for the understanding and optimization of the focusing.     

固化pH梯度毛细管等电聚焦整体柱的制备及在蛋白质等电点分析中的应用

通过聚合物原位聚合反应，制备了部分填充的毛细管整体柱。pH 3~10的载体两性电解质被固化在该毛细管整体柱上。在引入八通进样阀、三通阀和四通连接单元的基础上，构建了适用于固化pH梯度毛细管等电聚焦整体柱（M-IPG）的平台。在蛋白质药物测定过程中，用M-IPG柱和羟丙基纤维素（HPC）涂层毛细管柱同时对曲托珠单抗和依那西谱的等电点进行了测定。结果表明，两种等电聚焦柱都能够同时分离混合蛋白质样品并测定蛋白质类药物中单抗和融合蛋白质的等电点（pI），M-IPG柱所测的pI值与HPC涂层毛细管柱测定的结果基本一致，表明了该柱在进一步构建多维分离平台进行蛋白质组学研究方面的潜力。     

Biotinylation of TiO(2) nanoparticles and their conjugation with streptavidin

Photoactive TiO(2) can be used to mediate a variety of disinfection processes. It was postulated that TiO(2) particles could be directed to specific targets of interest using biotin/streptavidin linkages. Biotinylated TiO(2) nanoparticles (anatase) were obtained by treating TiO(2) nanoparticles with 3-aminopropyltriethoxysilane (APTS) in anhydrous DMSO, followed by reaction with N-hydroxysuccinimidobiotin. 29Si CP-MAS NMR, 13C CP-MAS NMR, and FTIR spectra showed that biotin was covalently bound to the TiO(2) surface. Transmission electron microscopy (TEM) demonstrated that prolonging the silanization reaction times led to increasingly thick silsesquioxane coating layers of up to approximately 10 nm. The specific surface area (SSA) of the TiO2 particles decreased from 16 m(2) g(-1) before treatment to 9.1 m(2) g(-1) after aminosilanization and to 8.4 m(2) g(-)1 after biotinylation, as measured by nitrogen adsorption. Amino surfaces modified for 4, 16, and 26 h had total amino group densities ranging from 2.9 to 26 to 66 nm(-2), respectively, whereas accessible surface amino group densities ranged from 2.7 to 10 to 17 nm(-2) as shown from nitrogen adsorption, polyelectrolyte titration, conductometric titration, and biotin assays. Not all the amino groups were accessible for biotinylation: the densities of active biotin were found to be 2.1, 7.0, and 11.5 nm(-2). The ability of the attached biotin to bind to streptavidin was demonstrated by confocal microscopy with the use of fluorescently labeled streptavidin-FITC. Although streptavidin was readily able to bind to biotinylated TiO(2) particles, it did not act as a strong flocculating agent for the biotinylated TiO2 particles. The implications of these observations, with respect to particle accessibility to tethered streptavidin, are discussed.