A modified method for determining protein binding capacity of plant polyphenolics using radiolabelled protein

A modified radiochemical protein binding method for determining the protein binding capacity of plant polyphenolics (tannins) is described. Purified tannin or unfractionated plant extracts were immobilised on filter paper discs and incubated with the 125I-labelled bovine serum albumin. Protein bound to the disc was proportional to the amount of tannin applied to the disc, although at high concentrations of polyphenolics the discs became saturated and the relationship was no longer applicable. The method was validated using purified procyanidin from Sorghum grain and has been applied to crude polyphenolic extracts from maple, white oak, black oak, walnut and tulip poplar leaves. Specific chemical assays for the determination of proanthocyanidins (acid butanol method) and hydrolysable tannins (modified potassium iodate method) were employed to validate the new protein binding method with the complex plant extracts.     

Competitive protein binding assay of progesterone without chromatography

A competitive protein binding assay for the measurement of progesterone in human plasma without chromatographic separation of steroids and recovery evaluation in individual samples is described. It is based on the specificity of the progesterone binding plasma protein (PBP) of the pregnant guinea pig. A dried petroleum ether extract of plasma was incubated with ³H-progesterone and 1600 fold diluted pregnant guinea pig plasma. Bound radioactivity was measured with a dextran coated charcoal suspension technique. Plasma progesterone concentration was obtained by comparison with a standard curve and correction for extraction separately measured for each batch of petroleum ether. The sensitivity was 100 pg. Recovery experiments for progesterone and competing steroids added to plasma respectively showed the accuracy and the specificity of the method. However comparison of the results from assays with and without chromatographic separation of steroids, showed that in the latter-case the specificity was good only for plasmas containing more than 1ng/ml of progesterone. Concentration of progesterone in plasma from men was 0.46±0.14 ng/ml (mean ± S.D) and from post menopausal women 0.30± 0.13 ng/ml.Between days 1 and 13 and days 16 and 22 of the normal menstrualcycle the concentrations were respectively 0.81 ± 0.38 and 12.50 ± 2.96 ng/ml. The variations of the progesterone concentration during pregnancy are also shown.     

Quantitative reappraisal of general expressions for multivalent protein binding in subunit-exchange chromatography

Quantitative expressions have been derived for bivalent equilibria with immobilized ligand systems and for the equilibria for an immobilized protein whose self-association is modified by binding with a soluble ligand, as analyzed by affinity chromatography. These general expressions have been applied in a reexamination of multivalency in the affinity chromatography of antibodies, as reported by Eilat and Chaiken (Biochemistry 18 (1979) 790) and also to studies of neurophysin-peptide hormone interactions using glass matrices reported by Swaisgood and Chaiken (Biochemistry 25 (1986) 4148).     

High-performance affinity chromatography: a powerful tool for studying serum protein binding

High-performance affinity chromatography (HPAC) is a method in which a biologically-related ligand is used as a stationary phase in an HPLC system. This approach is a powerful means for selectively isolating or quantitating agents in complex samples, but it can also be employed to study the interactions of biological systems. In recent years there have been numerous reports in which HPAC has been used to examine the interactions of drugs, hormones and other substances with serum proteins. This review discusses how HPAC has been used in such work. Particular attention is given to the techniques of zonal elution and frontal analysis. Various applications are provided for these techniques, along with a list of factors that need to be considered in their optimization and use. New approaches based on band-broadening studies and rapid immunoextraction are also discussed.     

A model membrane protein for binding volatile anesthetics

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.     

Affinity chromatography of a sequence-specific DNA binding protein using Teflon-linked oligonucleotides

An affinity matrix was constructed by synthesis of a DNA oligonucleotide on a Teflon fiber support followed by deblocking and hybridization of the complementary strand. It was used to purify a sequence-specific binding protein at least 100-fold to near homogeneity. This matrix is easily fabricated on an automated DNA synthesizer, contains high levels of attached DNA, and has superior mechanical properties. It should be generally useful for affinity chromatography of DNA binding proteins.     

Calcium binding capacity and calcium sensitive protein content in denervated frog muscles

Total and ionic calcium content, calcium binding capacity of sarcoplasmic proteins and calcium insensitive proteins were examined in atrophying leg muscles of frog after 1-5 months period of denervation. Different muscles showed different levels of atrophy and the total calcium content varied with reference to the type of muscle. Ionic calcium levels doubled in the gastrocnemius muscle after three months denervation. Calcium binding capacity of proteins and calcium insensitive proteins decreased rapidly up to four months after denervation in the gastrocnemius muscle. However no significant changes in the levels of calcium binding capacity and calcium insensitive proteins were found with reference to the type of muscle. Since total calcium content remains constant and wet muscle mass (expressed as atrophy) decreased markedly, an apparent increase in calcium concentration occurs in each muscle on denervation.     

Zinc binding reverses the calcium-induced arachidonic acid-binding capacity of the S100A8/A9 protein complex

Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 calciums per EF-hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn2+, Cu2+, and Mg2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn2+ or Cu2+ in the presence of calcium, whereas Mg2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein-protein interaction assay. Fluorescence measurements gave evidence that both Zn2+ and Cu2+ induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.     

A competitive protein binding assay for allopurinol and oxipurinol

A competitive protein binding assay for allopurinol or oxipurinol has been developed based on the tight binding of these drugs to reduced xanthine oxidase. Free drug is separated from that bound to xanthine oxidase by absorption with dextran-albumin coated charcoal. This assay can detect as little as 0.1 μm allopurinol or oxipurinol in water, serum, plasma, or urine. Competitive analogs such as hypoxanthine, xanthine, and uric acid require concentrations 100- to 1000-fold greater than those of allopurinol or oxipurinol to cause significant interference with the assay. This assay is simple and rapid with the ability to assay 20–30 samples within 2 h. Measurement of oxipurinol levels in clinical samples shows good correlation with published results using more complex analytical techniques.     

A new method for determining the heat capacity change for protein folding

In order to use results from calorimetry or thermal unfolding curves to estimate the free energy change for protein unfolding at 25 degrees C, it is necessary to know the change in heat capacity for unfolding, delta Cp. We describe a new method for measuring delta Cp which is based on results from urea and thermal unfolding curves but does not require a calorimeter. We find that delta Cp = 1650 +/- 200 cal/(deg.mol) for the unfolding of ribonuclease T1 and that delta Cp = 2200 +/- 300 cal/(deg.mol) for the unfolding of ribonuclease A.     

A simple method for measurement of the haemoglobin binding capacity of canine haptoglobin

The potential of a series of related compounds to induce haemolytic anaemia in dogs highlighted the need for a reliable and sensitive technique to identify changes in plasma haptoglobin concentration. An indirect method established for human samples was adapted for use with canine plasma. This method measures the haemoglobin binding capacity of plasma, which is directly proportional to the functional haptoglobin concentration. The efficacy of this technique was investigated on samples taken from Beagle dogs which had previously received small quantities of water intravenously. A substantial reduction in haemoglobin binding capacity was recorded before other haematological parameters were significantly affected. It was concluded that measurement of haemoglobin binding capacity by this method provides a valid reflection of haptoglobin status in the dog.     

Affinity chromatography for protein isolation.

Thousands of reports concerning protein purification have appeared in the past year, and over 150 of these involved, at least in part, the affinity chromatography process. Immobilized membrane affinity chromatography, temperature-programmed elution, and centrifugal affinity chromatography are among the most significant new techniques amid the myriads of applications in this mature field.     

A binding protein from rat nerve

Leucine has been found to bind competitively to a soluble protein (molecular weight 97,000 daltons) from rat sciatic nerve under certain experimental conditions to form a high molecular weight aggregate (MW greater than 302,000). Kinetic study showed that the equilibrium constant for leucine-binding is 1.33 X 10(4) l/m and the rate constants for binding and unbinding are k1 = 0.424 l/m/sec and k-1 = 3.18 X 10(5) sec-1 respectively. The binding reaction is accompanied by an endothermic enthalpy change of 5,000 cal/mole and the favorable equilibrium appears to be due to the large positive (35.3 eu) entropy of binding. L-Proline, thymidine, and succinic acid were also found to bind, non-competitively with leucine, to proteins in the same fraction. Binding of those compounds and leucine was enhanced by the presence of Mg2+. Rat muscle and plasma proteins did not significantly bind leucine under these experimental conditions. The presence of this binding protein in rat nerve suggests an additional mechanism in the metabolism and in the transport of amino acids for incorporation into a protein structure in nerve.     

Influence of endocytosis-stimulating hormones on the protein binding capacity of the cell membrane

Histamine and serotonin enhanced the adsorption of fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) to the membrane of Tetrahymena in a statistically significant and biologically relevant degree, while the similar action of insulin and epinephrine was not significant in either respect. The degree of hormonal influence on BSA-binding was similar to that exerted by the same hormones on the phagocytosis of protozoa in earlier studies. This supports the hypothetical implication that enhancement of protein adsorption to the cell membrane is an important element in the endocytosis stimulating action of histamine and serotonin.     

A rapid method for determining dynamic binding capacity of resins for the purification of proteins

Biolayer interferometry is a novel method for quantifying macromolecules, such as proteins, in solution. The presence of other, non-binding molecules does not interfere with quantification, which allows one to measure the concentration of the molecule of interest in a crude mixture. Here we apply this method to determining the dynamic binding capacity of affinity resins.     

Design and characterization of a microfluidic packed bed system for protein breakthrough and dynamic binding capacity determination

A 1.5 μL ion exchange chromatography column to accommodate resins used for biopharmaceutical processing has been designed to produce breakthrough curves and to quantify dynamic and maximum protein binding capacities. Channels within a glass chip were fabricated using photolithography and isotropic etching. The design includes a 1 cm long microfluidic column in which compressible, polydispersed porous agarose beads (70 μm mean diameter) were packed using a keystone method where particles aggregate in a narrow channel. The depth of the column is such that two bead layers exist. The fabrication technique used forms Cartesian geometries as opposed to circular cross sections found in standard columns. The voidage was therefore higher than standard values when measured by 3D confocal microscopy. In conjunction with microscopic techniques, the column allows visualization of events within the bed such as adsorption profiles that would otherwise be difficult to observe. In this work, the binding of fluorescently labeled protein during isocratic loading was used to generate breakthrough from the microcolumn. Useful breakthrough curves were achieved using mobile phase velocities from 60 to 270 cm h^?1. Calculated dynamic binding capacities were compared well with previously published data on conventional scale columns. The microfluidic chromatography column described here thus allows study of process scale chromatography behavior at scales 20,000 times smaller than in current practice. The work described in this article is representative of the proof of principle of a potentially powerful tool for the generation of microfluidic process bed data for the biopharmaceutical industry. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009