Near-infrared fluorescence lifetime assay for serum glucose based on allophycocyanin-labeled concanavalin A

We describe an assay scheme for glucose based on fluorescence resonance energy transfer (FRET) between concanavalin A (con A), labeled with the near-infrared fluorescent protein allophycocyanin (APC) as donor, and dextran labeled with malachite green (MG) as acceptor. Glucose competitively displaces dextran-MG and leads to reduction in FRET, assessed by time-domain fluorescence lifetime measurements using time-correlated single-photon counting. The assay is operative in the glucose concentration range 2.5-30 mM, and therefore suitable for use in monitoring diabetes control. Albumin and serum inhibit FRET but the interference can be prevented by removal of high molecular weight substances by membrane filters. APC shows promise for incorporation in an implanted glucose sensor which can be interrogated from outside the body.     

Coupling of concanavalin A to cellulose hollow fibers for use in glucose affinity sensor

This article describes a method for the chemical immobilization of concanavalin A (Con A) on the inside wall of a single hollow cellulose fiber for use in glucose affinity sensor. Periodate oxidation of cellulose fiber followed by a spacer for Con A attachment was deemed to be the most optimal procedure for achieving the highest sensitivity of the sensor without compromising its physical integrity. The effects of variables like the duration of periodate oxidation and its concentration and pH of the spacer coupling step and its duration have been examined. The mechanical strength of the hollow fiber as well as its permeability to the analyte (glucose) have been evaluated prior to and after Con A coupling process.It has been demonstrated that Con A bound hollow fiber prepared according to the procedure outlined here can be successfully used to construct glucose affinity sensor for operation in the physiological range of glucose concentrations.     

A sensitive competency assay for concanavalin A binding

A standardized assay method has been developed for assessing the binding capacity of derivitized concanavalin A (con A). The method takes advantage of the fact that concanavalin A binds to Sephadex G-75 in an α-methyl-d-mannopyranoside-inhibitable manner. Using this binding assay we have found that α-methyl-d-mannopyranoside inhibition of [¹²⁵I]concanavalin A to Sephadex is greater than 95%. In addition, this sugar is able to remove more than 99% of [¹²⁵I]concanavalin A previously bound to Sephadex. Competition studies will allow the use of this procedure to characterize any con A sample under the exact conditions used for cell-binding studies.     

Isolation of concanavalin a by affinity precipitation

Summary Concanvalin A (Con A) was isolated from a crude extract of jack beans by affinity precipitation. p-Aminophenyl--D-glucopyranoside was coupled to the polymer Eudragit S-100. Affinity binding took place at pH 7.5 and precipitation was initiated by lowering pH to 5.2. The lectin was eluted by dissolving the polymer in the presence of 0.2 M methyl--D-mannopyranoside and then precipitating the polymer only. The polymer-ligand was recycled and used five times.     

The assessment of potentially interfering metabolites and dietary components in blood using an osmotic glucose sensor based on the concanavalin A-dextran affinity assay

Continuous surveillance of blood glucose is a prerogative of maintaining a tight glycaemic control in people suffering from diabetes mellitus. Implantable sensor technology offers the potential of conducting direct long term continuous glucose measurements, but current size restrictions and operational challenges have limited their applications. The osmotic sensor utilises diffusion to create a hydrostatic pressure that is independent of sensor operation and power consumption. This permits ultra-low power architectures to be realized with a minimal start-up time in a package suitable for miniaturization. In contrast, osmotic sensors suffer from the inability of their membranes to discriminate between different constituents in blood or the interstitial fluid that are of comparable size to glucose. By implementing an affinity assay based on the competitive bonding between concanavalin A and dextran, the selectivity of the membrane can be transferred to the glucose specific recognition of the affinity assay. The osmotic effect from the physiological levels of several key metabolites and nutritional components has been addressed identifying in particular ethanol, lactate and amino acids as potential interfering constituents. Both ascorbic acid and mannose would have a normal physiological concentration that is too low to be detected. The studies shows that an osmotic glucose sensor equipped with the con A-dextran affinity assay, is able to filter out potential interfering constituents present in blood, plasma and the interstitial fluid yet retaining a pressure that is proportional to glucose only.     

Effect of the conformation of concanavalin A on its affinity for manganous ion

The stoichiometry of Mn2+ binding to concanavalin A at pH 6.4-7 which had been established in two independent studies [J.A. Sophianopoulos, A.J. Sophianopoulos, and W.C. MacMahon (1983) Arch. Biochem. Biophys. 223, 350-359; D.J. Christie, G.R. Munske, and J.A. Magnuson (1979) Biochemistry 18, 4638-4644] was challenged [C.F. Brewer, R.D. Brown, III, and S.H. Koenig (1983) Biochemistry 22, 3691-3702] on grounds of possible experimental errors. Additional evidence is presented in this study in support of the previous finding that at pH 6.4 only one Mn2+ binds per concanavalin A monomer of Mr 25,550. Also, evidence is presented showing that the results of Sophianopoulos et al. could not have been due to contamination by Ca2+. A comparison is made of the results in the three studies cited above which indicates that the concanavalin A used by Brewer et al. had decreased affinity for Mn2+ and it contained an appreciable fraction of concanavalin A incompetent of binding saccharides.     

Identification of a breast tumor-associated orosomucoid by concanavalin A affinity chromatography.

Con A-Sepharose affinity chromatography was utilized to examine the glycoproteins in phosphosaline extracts of normal and breast tumor tissues and breast patient sera. In extracts of normal breast tissue, normal sera and patient sera, all glycoproteins were eluted from the Con A-Sepharose with a linear gradient of 0.0-0.5 M alpha-methylmannose. Using breast tumor extracts, a glycoprotein peak which could not be eluted as with normal tissue extracts was observed. This tightly-binding peak could be eluted from the Con A-Sepharose with acetate buffer containing 1.0 M KCl. Polyacrylamide electrophoresis of this tightly-binding glycoprotein peak revealed one major glycoprotein and four minor glycoproteins. The major glycoprotein obtained from electrophoresis represented about 60% of the Con A-Sepharose tightly-binding protein and reacted with antiserum to human orosomucoid (alpha 1-acid glycoprotein). All glycoproteins isolated from tumor tissue extracts appeared to represent normal serum constituents as they were retained on an immunoadsorbent containing antibodies to normal serum proteins. The possible significance of the isolated tumor-associated orosomucoid is discussed.     

Electrophoretic desorption of concanavalin a from an affinity matrix: A rapid method for the preparation of carbohydrate free concanavalin A

Summary Electrophoretic desorption of concanavalin A from Sephadex G 50 is described. The method is simple, rapid, eliminates dialysis and biologically active carbohydrate free lectin is obtained within an hour.     

A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.     

The purification of human serum 1 -antitrypsin by affinity chromatography on concanavalin A

α₁-Antitrypsin (α₁-AT) has been isolated from human serum by a two-step procedure which involves chromatography on DEAE-Sephadex followed by affinity chromatography on insolubilized concanavalin A. This protein appeared to be homogeneous when examined by electrophoresis on cellulose acetate and double immunodiffusion; minor contaminants, however, were detected by gel electrophoresis and immunoelectrophoresis. This procedure is readily adaptable to the large-scale purification of α₁-AT and should facilitate further studies on the physicochemical and biological properties of α₁-AT and its genetic variants.     

The affinity of concanavalin A and wheat germ agglutinin for components of the muscle spindle.

Sections through the soleus muscle of the rat were incubated with concanavalin A (Con A) or wheat germ agglutinin (WGA) conjugated to fluorescein isothiocyanate. Binding of these lectins to structures which comprise the muscle spindle was studied by fluorescence microscopy. The distribution of the lectins was heaviest in the outer capsule of the spindle and at the surface of intrafusal muscle fibres. The periaxial space in the equatorial region of spindles was unlabelled except in the immediate vicinity of the axial bundle. Binding by Con A was more extensive than by WGA, suggesting that more glucopyranoside units are accessible within the muscle spindle than are those of N-acetylglucosamine.     

Differential effects of concanavalin A and succinyl concanavalin A on the macromolecular events of platelet activation

Concanavalin A is capable of activating platelets in a concentration-dependent manner as judged by [14C]serotonin secretion from prelabeled platelets. In contrast, succinyl concanavalin A does not induce platelet secretion. Concanavalin A treatment also results in a number of alterations in platelet macromolecules which are presumably associated with the process of platelet activation. These include the phosphorylation of 20 and 47 kDa platelet proteins, the increased polymerization and association of new proteins with the platelet cytoskeleton and the association of the platelet membrane glycoprotein IIb/III complex with the platelet cytoskeleton. Succinyl concanavalin A treatment results in none of these macromolecular events. This difference is observed despite the demonstration that both lectins bind to the platelet surface. Gel overlay experiments also indicate that concanavalin A and succinyl concanavalin A bind to the same receptors. These differences in the biological effects of concanavalin A and succinyl concanavalin A on platelets may be due to decreased receptor crosslinking by the succinylated derivative. The formation of multiple linked interactions between surface receptors may be an important event in the activation of platelets by concanavalin A.     

Colorimetric determination of fructose for the high-throughput microtiter plate assay of glucose isomerase

A colorimetric method for the reducing monosaccharide determination is optimized for the assay of glucose isomerase, which converts glucose (Glc) to fructose (Fru). Test solution was mixed with 20-fold volume of the 50 mM Na₂SiO₃, 600 mM Na₂MoO₄, and 0.95 M HCl aqueous solution (pH 4.5), in which a yellow molybdosilicate species was formed. The mixture was kept at 70 °C for 30 min. Test solution containing 10 mM level Fru gave a remarkable blue reaction mixture, in which the Mo(VI) species was reduced by Fru to form a blue molybdosilicate species. The blueness increased with the Fru concentration. Glc cannot render the reaction mixture blue as strong as Fru. Thus, the colorimetric method can be used advantageously for the determination of 10 mM level Fru in the Glc isomerase reaction mixture, even in the presence of 100 mM level Glc, and has been applied successfully to the microtiter plate assay of the enzyme.     

Glycan uniformity within molecular variants of transferrin with distinct affinity for concanavalin A

Human serum Transferrin has been fractionated on concanavalin A-Sepharose column into three fractions: Con A-non reactive (5 %), Con A-weakly reactive (30 %) and Con A-reactive (65 %). Each Con A-affinity transferrin variant contains a pair of identical oligosaccharide units either tri-branched in the Con A-non reactive variant or two-branched in the Con A-weakly reactive and Con A-reactive transferrin. This is an additional support to our proposal that glycosylation occurs uniformly along each polypeptide chain yielding identical oligosaccharide units at each glycosylated site.     

Purification of the glycoprotein allergen Ag7 from mugwort pollen by concanavalin A affinity chromatography.

Two affinity columns comprising immobilized concanavalin A (Con A), Con A-Sepharose and Con A-XP3507, were evaluated for their purifying ability for the glycoprotein allergen Ag7 from a partially purified extract of mugwort pollen. The most pronounced difference between the two columns was the nature of their nonspecific interactions; hydrophobic interactions were dominant with Con A-XP3507, whereas ionic interactions were dominant with Con A-Sepharose. Both Con A-columns were effective for purifying Ag7 with a recovery of 50% after specific elution with displacing sugars. The inclusion of 1.0 M NaCl and 20% ethylene glycol in the elution medium was useful for desorbing nonspecifically bound material, prior to specific elution of adsorbed Ag7 in the presence of the displacing sugars, alpha-methyl glucoside and alpha-methyl mannoside. The most efficient purification of Ag7 was achieved with Con A-Sepharose at room temperature rather than at 4 degrees C. Affinity chromatography with Con A-XP3507 resulted in a slightly more contaminated product (purity 54%) than with Con A-Sepharose (purity 64%).