Determination of protein covalently bound to agarose supports using bicinchoninic acid

A method using bicinchoninic acid (BCA) for the direct determination of protein covalently bound to agarose is described. The method involves the preparation of a standard curve using solubilized protein, the determination of the slurry concentration of the gel sample, the colorimetric assay of the gel slurry using BCA in a serum separator device, and the calculation of the amount of protein bound to the gel using the standard curve of the solubilized protein. The use of the BCA method for direct estimation of immobilized protein agrees well with the "balance" method which depends upon the measurement of protein depletion and yields highly reproducible results with a variety of coupling chemistries commonly used with agarose beads. The use of commercially available serum filters provides for a fast, less than 1 h, and convenient analytical format.     

Antibody response to bacterial antigens covalently bound to biodegradable polymerized serum albumin beads

Model vaccines have been made by covalently linking Clostridium botulinum type D toxin and Klebsiella pneumoniae capsular polysaccharide antigen to polymerized rabbit serum albumin beads. When injected into rabbits these bead vaccines induced an enhanced production of specific humoral antibody without causing adverse reactions. The adjuvant effect is due to a slow release from the bead structure and offers an alternative to oil emulsions and mineral salt adsorbents.     

Antibody response to bacterial antigens covalently bound to biodegradable polymerized serum albumin beads

Model vaccines have been made by covalently linking Clostridium botulinum type D toxin and Klebsiella pneumoniae capsular polysaccharide antigen to polymerized rabbit serum albumin beads. When injected into rabbits these bead vaccines induced an enhanced production of specific humoral antibody without causing adverse reactions. The adjuvant effect is due to a slow release from the bead structure and offers an alternative to oil emulsions and mineral salt adsorbents.     

A rapid, accurate, nonradioactive method for quantitating RNA on agarose gels.

In order to study quantitative gene expression with Northern blots, it is important to have an internal standard that can be used to verify even loading or to correct for uneven loading between lanes. In this study it is shown that two-dimensional quantitation of ethidium bromide-intercalated 28S rRNA fluorescence can be used for such standardization. It was found that the film response of the fluorescence was linear with respect to total loaded RNA in the range of 2.5-12.5 micrograms RNA under the conditions used, after which the linear relationship falls off. This method eliminates the use of radiation for internal standardization of Northern blots.     

Hydroxamate assay quantitation of ligands covalently bound to affinity chromatography gels

Affinity chromatography which utilizes specific biological interactions in the purification or analysis of a variety of biochemical systems is an exceedingly useful method. The technique was initially limited to the use of immunoadsorbants (1) and since has been broadened in scope largely by the efforts of Cuatrecases, Anfinsen and colleagues (2,3). In principle, a specific ligand interacting with a particular substance, usually a macromolecule, is covalently bound to an insoluble support. Substances with no affinity for the ligand will pass unretarded through a column of the bound support; whereas, the interacting materials will be retarded. The methods for preparing a variety of affinity-chromatographic systems are now readily available (2,3). However, a quantitative measure of the covalently bound ligand in many instances depends on the use of radioactively labelled ligands, access to an automatic amino acid analyser, or the subtraction of the amount of ligand recovered in washings after the binding reaction. The latter method is often inaccurate and the former methods may not be practical for all laboratories.We have employed successfully the hydroxamate assay (4,5) for measuring biotin linked through an amide bond to the ω amino group of a 3,3′-diaminodipropylamine substituted agarose gel. This method with individual modifications should be of general use in measuring ligands bound to solid supports through amide, ester, or thioester bonds.     

Application of spectrophotometric methods to the determination of protein bound to agarose beads.

The proteolytic activities of α-chymotrypsin, trypsin, pepsin, bromelain, and an extract from germinating pumpkin seeds were determined by their ability to effect the release of 1-anilino-8-naphthalenesulfonate bound to internal hydrophobic sites in intact protein substrates resulting in a decline in fluorescence. Casein, glyceraldehyde-3-P dehydrogenase, urease, catalase, pumpkin seed globulin, and bovine serum albumin enhanced the fluorescence of 1-anilino-8-naphthalenesulfonate sufficiently to be used as proteolytic substrates in this assay procedure. The activity of 1 μg chymotrypsin or trypsin and 100 ng pepsin could be easily detected by this method within 4 to 8 min depending upon the protein substrate. The digestive enzymes and bromelain exhibited activity against most if not all six of the protein substrates used. In contrast, the extract from germinating pumpkin seeds exhibited significant activity only against pumpkin seed globulin, with maximal activity at pH 7.4. Compared with the assay method for proteolytic activity utilizing ninhydrin analysis of the reaction products, this method was at least 10 times more rapid and gave significant detectable activity with much lower quantities of proteolytic enzyme.     

Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

A protein covalently bound to ColE1 DNA

ColE1 DNA was isolated from Escherichia coli as a relaxation complex of supercoiled DNA and proteins. Treatment of the complex with either protein-denaturing agents (SDS, phenol etc.) or proteolytic enzymes converted the supercoiled DNA to an open-circular form (relaxation). The relaxation complex was separately labelled in vivo with [3H]Leu or [14C]Leu, [35S]Met or (32P)phosphate and extensively purified. Complete hydrolysis of the relaxed complex with DNase I and P1 nuclease produced a 36-kDa protein which, we believe, is covalently bound to ColE1 DNA. On the other hand, the relaxed complex was treated with tosylphenylalanylchloromethane-treated-trypsin and the DNA-peptide(s) produced was (were) isolated and digested with the nucleases as above. The resulting nucleotidylpeptide(s) was (were) isolated by DEAE-Sephadex chromatography. The only 5'-dCMP was released from the nucleotidylpeptide(s) by snake venom phosphodiesterase treatment. O-Phosphoserine was found in acid hydrolysates of the DNA-peptide(s). We suggest that in the relaxation event the 36-kDa protein becomes covalently linked to ColE1 DNA via a phosphodiester bond between dC and the serine residue.     

Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads

In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.     

A simple method for counting antibody bound labelled steroids.

A simple method is described for the counting of tritiated antibody-bound steroid after acidification and extraction into a toluene based scintillant. The resulting count rate is stable and quenching is minimal. The method give improved count rates when compared with methods employing scintillants containing methanol, dioxane, or Triton and compares favourably with methods involving pre-heating of antibody-bound steroid or extraction of free steroids after the addition of ammonium sulphate. A specific antibody for estradiol has been used to illustrate the application of the technique to antibody titration curves and standard curves.     

Crystal structure of trioxacarcin A covalently bound to DNA

We report a crystal structure that shows an antibiotic that extracts a nucleobase from a DNA molecule ‘caught in the act’ after forming a covalent bond but before departing with the base. The structure of trioxacarcin A covalently bound to double-stranded d(AACCGGTT) was determined to 1.78 Å resolution by MAD phasing employing brominated oligonucleotides. The DNA–drug complex has a unique structure that combines alkylation (at the N7 position of a guanine), intercalation (on the 3′-side of the alkylated guanine), and base flip-out. An antibiotic-induced flipping-out of a single, nonterminal nucleobase from a DNA duplex was observed for the first time in a crystal structure.     

Plasma membrane glycoproteins covalently bound to silica beads as a model for molecular studies of cell-cell interactions in culture

In previous studies, we have shown that plasma membrane glycoproteins are of major importance in the density-dependent regulation of growth of normal diploid fibroblasts. Due to the hydrophobic portions of these molecules, functional studies in cell culture are often difficult to perform and to interpret. Specifically, the addition of these molecules in soluble form to cell culture, after depletion of detergents needed for their solubilization, leads to aggregation and internalization. Therefore, we developed a method for the covalent immobilization of the solubilized plasma membrane proteins to derivatized silica beads for further investigations on the molecular nature of the active molecules. The addition of immobilized plasma membrane glycoproteins to sparsely seeded human fibroblasts resulted in cellular reactions similar to those found in confluent cell cultures (strongly reduced cell proliferation; high collagen type III synthesis). The method consists in the derivatization of silica beads (Lichrosphere Si 500, 10 microns) with isothiocyanatopropyltriethoxysilane. Amino-groups react with the SCN group under physiological conditions, resulting in a stable linkage of amino-group bearing molecules with the silica beads. Due to the easy handling of the silica beads (e.g. washing by short centrifugation steps), the mild coupling conditions, and the stable bondings this system is highly suited for functional studies of molecules involved in cell-cell interactions.     

A disposable bio-nano-chip using agarose beads for high performance immunoassays

This article reports on the fabrication of a disposable bio-nano-chip (BNC), a microfluidic device composed of polydimethylsiloxane (PDMS) and thiolene-based optical epoxy which is both cost-effective and suitable for high performance immunoassays. A novel room temperature (RT) bonding technique was utilized so as to achieve irreversible covalent bonding between PDMS and thiolene-based epoxy layers, while at the same time being compatible with the insertion of agarose bead sensors, selectively arranged in an array of pyramidal microcavities replicated in the thiolene thin film layer. In the sealed device, the bead-supporting epoxy film is sandwiched between two PDMS layers comprising of fluidic injection and drain channels. The agarose bead sensors used in the device are sensitized with anti-C-reactive protein (CRP) antibody, and a fluorescent sandwich-type immunoassay was run to characterize the performance of this device. Computational fluid dynamics (CFD) was used based on the device specifications to model the bead penetration. Experimental data revealed analyte penetration of the immunocomplex to 100 μm into the 280 μm diameter agarose beads, which correlated well with the simulation. A dose-response curve was obtained and the linear dynamic range of the assay was established over 1 ng/mL to 50 ng/mL with a limit of detection less than 1 ng/mL.     

Interaction of thyroid peroxidase with concanavalin A covalently coupled to agarose.

We have investigated the interaction between concanavalin A-agarose (Con A-agarose) and thyroid peroxidase, an integral membrane protein found in the 105,000 X g, 1-h particulate fraction of thyroid tissue. An intact form of porcine thyroid peroxidase was obtained by solubilization with the nonionic detergent Triton X-100 and two fragmented, hydrophilic forms of the enzyme were prepared by trypsin treatment of the membrane. The three types of thyroid peroxidase bind to Con A-agarose and can be eluted with alpha-methyl-D-mannoside. The alpha-methyl-D-mannoside eluate of the most purified thyroid peroxidase preparation has been analyzed by polyacrylamide gel electrophoresis. Peroxidase activity corresponds with a glycoprotein band. The binding of thyroid peroxidase to Con A-agarose can be inhibited by sugars in the following order: alpha-methyl-D-mannoside greater than D-mannose greater than alpha-methyl-D-glucoside greater than D-glucose greater than D-galactose. This order of specificity is typical of Con A-sugar interactions. Furthermore, inactivation of the carbohydrate binding site of Con A by demetallization greatly reduces the extent of thyroid peroxidase binding. Reactivation of the carbohydrate binding site by the addition of Ca2+ and Mn2+ to demetallized Con A-agarose restores thyroid peroxidase binding. These and other experiments suggest that htyroid peroxidase is, like several other peroxidases, a glycoprotein. In addition, the interaction between thyroid peroxidase and Con A-agarose may provide a new purification tool for thyroid peroxidase.     

A simple method for recovering DNA from agarose gels using glass fibre filters

A simple and reproducible procedure for the recovery of plasmid DNA is described. The method was standardised for the purification of plasmids from Gluconobacter oxydans ATCC9937. The protocol is based on the use of glass microfibre filter paper for entrapment of DNA and its subsequent recovery by an elution buffer. The method precludes the use of phenol and butanol for the removal of proteins and ethidium bromide respectively, therefore, making the procedure inexpensive and gentle.