Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.      

A colorimetric assay specific for gamma-carboxyglutamic acid-containing proteins: its utility in protein purification procedures

A colorimetric method for the detection of gamma-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without gamma-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by gamma-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the gamma-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second gamma-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 microM for the gamma-carboxyglutamic acid-containing protein bone Gla protein and up to 27 microM for the amino acid. The detection limit is down to 1 microgram of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for gamma-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.     

发酵黄芪的乳酸菌的驯化及其与黄芪相互作用研究

目的驯化能发酵黄芪的优良乳酸菌并研究其与黄芪的相互作用,为开发新型饲料添加剂做准备。方法选取能厌氧发酵黄芪药液的优良乳酸菌,通过在添加黄芪药液的发酵培养基中反复传代对其进行驯化,并用蒽酮-硫酸法测定发酵前后粗多糖的含量,用比浊法测定菌体浓度,并用单因素法研究了最佳黄芪添加量和最佳接种量,初步研究驯化菌株与黄芪药液的相互作用。结果经过6次传代驯化后,驯化菌株发酵黄芪药液后可使其粗多糖得率提高111．9％,而黄芪药液能促进驯化菌株的增殖,可使菌体浓度提高251％。结论驯化菌株与黄芪药液发生了良性的相互作用,有可能开发出新型饲料添加剂。     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Effects of cations,sugars, detergents,sulfhydryl compounds,and cryoprotective agents on the colorimetric determination of N-acetylglucosamine by the method of reissig

The monovalent cations, K⁺ and Na⁺, and the detergents, Triton X-100, Hyamine 2389, and sodium dodecyl sulfate, as well as the sugars, sucrose and α-methylglucoside, do not interfere with the colorimetric determination of N-acetylglucosamine. On the other hand, divalent cations, cryoprotective agents, and sulfhydryl compounds do influence the test of Reissig. By order of increasing effectiveness in reducing color production we find, among the cations, Mg²⁺ < Ca²⁺ < Mn²⁺ ≤ Zn²⁺; among the cryoprotective agents, dimethylsulfoxide < polyethylene glycol < glycerol; and among the sulfhydryl compounds, mercaptoethanol < dithiothreitol < cysteine. Not every interfering agent exerts its effect through the same mechanism. Only the first step of the test of Reissig is impaired by the divalent ions and dimethylsulfoxide while both steps are affected by the other agents. By using the appropriate calibration curve it is possible to determine the real effect of any interfering agent on an enzyme whose activity is monitored by the test of Reissig. By doing so, we have been able to show that Ca²⁺ does have an effect on bull acrosomal hyaluronidase; it activates the enzyme.     

Light scattering corrections to linear dichroism spectroscopy for liposomes in shear flow using calcein fluorescence and modified Rayleigh-Gans-Debye-Mie scattering

The interpretation of data from absorbance spectroscopy experiments of liposomes in flow systems is often complicated by the fact that there is currently no easy way to account for scattering artefacts. This has proved particularly problematic for linear dichroism (LD) spectroscopy, which may be used to determine binding modes of small molecules, peptides and proteins to liposomes if we can extract the absorbance signal from the combined absorbance/scattering experiment. Equations for a modified Rayleigh-Gans-Debye (RGD) approximation to the turbidity (scattering) LD spectrum are available in the literature though have not been implemented. This review summarises the literature and shows how it can be implemented. The implementation proceeds by first determining volume loss that occurs when a spherical liposome is subjected to flow. Calcein fluorescence can be used for this purpose since at high concentrations (>?60 mM) it has low intensity fluorescence with maxima at 525 and 563 nm whereas at low concentrations (<1 mM) the fluorescence intensity is enhanced and the band shifts to 536 nm. The scattering calculation process yields the average axis ratios of the distorted liposome ellipsoids and extent of orientation of the liposomes in flow. The scattering calculations require methods to estimate liposome integrity, volume loss, and orientation when subjected to shear stresses under flow.     

Determination of proteins and sulfobetaine with the folin-phenol reagent

This paper describes a method for the quantitative analysis of solutions containing a mixture of proteins and sulfobetaine. In a preliminary step the proteins, which interfere with the detergent assay, are separated by precipitation with trichloroacetic acid (8%). The insoluble fraction, dissolved in NaOH (1.0 n), and the soluble fraction, containing the detergent, are treated with the Folin-Ciocalteu phenol reagent, essentially following the method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem.193, 265–275). The absorbance of the protein fraction is read, as usual at 750 nm, while that of the detergent solution is read at 342 nm. At this wavelength, sulfobetaine, treated with the Folin reagent, absorbs strongly, the absorbance being proportional to its concentration up to 1.5 mg/ml.     

On the chemical basis of the Lowry protein determination

The copper-catalyzed oxidation of peptides and proteins by phosphomolybdic/phosphotungstic acid (Folin phenol reagent) was studied with respect to redox stoichiometry of color formation and nature of the oxidation products. From peptides without reducing side chains two reducing equivalents were transferred under ideal conditions to Mo6+/W6+ for each unit of tetradentate copper complex with concomitant formation of an imino peptide. Tyrosine and tryptophan side chains contributed four additional reducing equivalents. Oxidation of proline-containing peptides was greatly impaired as judged from color formation due to the interference of the imino acid with complex formation. Reaction of the oxidized peptides with 2,4-dinitrophenyl (DNP)-hydrazine gave a peptide amine and the DNP-hydrazone of a 2-oxoacyl peptide. The oxidation products from tetraalanine were identified as dialanine amide and pyruvoylalanine DNP-hydrazone. From the time course of the development of the blue color on reduction of Folin reagent with tetraalanine it was inferred that the reaction consisted of an initial (less than 5 s) oxidation to a Cu3+ peptide complex followed by slow changes in absorbance, especially above 0.2 mM. Due to these complications the two-electron stoichiometry has to be considered only as a limiting case for peptide concentrations below 0.02 mM.     

Atomic force microscopy of tomato and sugar beet pectin molecules

Atomic force microscopy has been used to image the structure of pectin molecules isolated from unripe tomato and sugar beet tissue. The tomato pectin molecules were found to be extended stiff chains with a weight average contour length of L_W = 174 nm and a number average contour length of L_N = 132 nm (L_W/L_N = 1.32). A proportion of the pectin molecules (30%) were found to be branched structures. Chemical analysis of the sugar beet pectin extracts showed that the samples contained protein (8.6%). This protein proved difficult to remove and is believed to be covalently attached to the polysaccharide. Imaging of the extracted pectin revealed largely un-aggregated chains: a small fraction (33%) of which were extended stiff polysaccharide chains and a major fraction (67%) of which were of polysaccharide–protein complexes containing a single protein molecule attached to one end of the polysaccharide chains (‘tadpoles’). In addition the sample contained a small number of aggregated structures. The un-aggregated pectin molecules were found to be predominately linear structures with a small fraction (17%) of branched structures. The branched structures were all in the free polysaccharide fraction and no branched pectin chains were observed in the protein–polysaccharide complexes. Alkali treatment was found to remove the protein. For the alkali-treated, un-aggregated structures the average contour lengths were found to be L_W = 137 nm, L_N = 108 nm with L_W/L_N = 1.27. It is proposed that the ‘tadpole’ structures contribute to the unusual emulsifying properties of sugar beet pectin.     

Rodlike complexes of a polyelectrolyte (hyaluronan) and a protein (lysozyme) observed by SANS

We study by small-angle neutron scattering (SANS) the structure of hyaluronan -lysozyme complexes. Hyaluronan (HA) is a polysaccharide of 9 nm intrinsic persistence length that bears one negative charge per disaccharide monomer (M(mol) = 401.3 g/mol); two molecular weights, M(w) = 6000 and 500,000 Da were used. The pH was adjusted at 4.7 and 7.4 so that lysozyme has a global charge of +10 and +8, respectively. The lysozyme concentration was varied from 3 to 40 g/L at constant HA concentration (10 g/L). At low protein concentration, samples are monophasic, and SANS experiments reveal only fluctuations of concentration, although, at high protein concentration, clusters are observed by SANS in the dense phase of the diphasic samples. In between, close to the onset of the phase separation, a distinct original scattering is observed. It is characteristic of a rod-like shape, which could characterize "single" complexes involving one or a few polymer chains. For the large molecular weight (500,000), the rodlike rigid domains extend to much larger length scale than the persistence length of the HA chain alone in solution and the range of the SANS investigation. They can be described as a necklace of proteins attached along a backbone of diameter of one or a few HA chains. For the short chains (M(w) ≈ 6000), the rod length of the complexes is close to the chain contour length (～ 15 nm).     

Beta-Elimination and sulfite addition reaction of chondroitin sulfate peptidoglycan and the peptide structure of the linkage region.

Pronase digestion of bovine tracheal cartilage yielded acid mucopolysaccharide - peptide complexes which were fractionated by chromatography on Dowex 1(C1-). A major fraction was eluted with 1.5 M NaC1 and presumed to by chondroitin sulfate A-peptidoglycan by cellulose acetate electrophoresis. Alkaline beta-elimination and sulfite addition reaction of this fraction yielded cysteic acid-containing peptides, two of which were obtained in an homogeneous state. The sequence determination of these two made it possible to remodel their original structures as Leu-Pro-Ser-Gly-Glu-Gly-Pro-Glu and Leu-Pro-Ser-Gly-Glu, where the serine residues carried polysaccharide chains. Together with the reported data on the polysaccharide-protein linkage region, the present result suggests that the -Ser-Gly- sequence is a minimum requisite for the glycosylation of serine residues in the protein core of various proteoglycans.     

A colorimetric assay for determination of cell viability in algal cultures

In this work, we propose the determination of cell viability in algal cultures by using a colorimetric assay widely used for estimation of cell proliferation in animal cell cultures. The method is based on in vivo reduction by metabolically active cells of a tetrazolium compound (MTS=3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt) to a colored formazan, with maximal absorbance at 490 nm, that is released to the culture medium. For this purpose, we have tested two microalgae with high commercial value (Dunaliella and Spirulina) and two seaweeds with different morphology (Ulva and Gracilaria). Color development in this assay is directly proportional to the number of viable cells, to the incubation time in the presence of the assay solution, and to the incubation temperature. A direct significant correlation was found between algal photosynthesis rate and color development in all species used through this work. Moreover, the intensity of absorbance at 490 nm was significantly lower in stressed cells (e.g. in nutrient-limited cultures, in the presence of toxic substances, and in osmotically-stressed cultures). We conclude that cell viability of algal cultures can be rapidly and easily estimated through colorimetric determination of the reduction of MTS to formazan.     

Direct visualization of collagens and procollagens in polyacrylamide gels.

This report describes a procedure that results in the rapid visual identification of collagens and procollagens in polyacrylamide gels. The technique results in a pink stain for collagenous proteins and a blue stain for all other proteins. The color difference has been evaluated spectrophotometrically. The absorbance maxima for collagen-Coomassie blue R250 complexes in gels is 520–535 nm, and the maxima for all other protein-Coomassie blue R250 complexes that we tested is 550–560 nm. This technique will facilitate the identification of collagenous proteins in complex mixtures of proteins derived from cell membranes, whole cell extracts, conditioned media, and extracellular matrices. We use the technique to detect procollagens in human diploid fibroblast conditioned media. The technique is simple, relatively rapid, has utility for proteins extracted by a variety of methods, and is applicable to all polyacrylamide gel systems in general use.     

Assay for enzyme activity by following the absorbance change of pH-indicators

Based on the absorbance change of indicators with the concentration of hydrogen ion released from an enzyme-catalyzed reaction, a convenient colorimetric method was established for the assay of acidic phospholipase A₂ and glycogen phosphorylase b. Brilliant yellow and bromothymol blue were chosen as indicators for assays of acidic phospholipase A₂ and glycogen phosphorylase b by following the absorbance changes at 495 and 615 nm, respectively. The method is simple, sample-saving, sensitive and valid for a wide range of enzyme concentrations. It can be extended for assaying other enzymes catalyzing reactions with hydrogen ion concentration changes.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-D-glucosamine.

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-D-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-D-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

Colorimetric protein assays, such as the Coomassie blue G-250 dye-binding (Bradford) and bicinchoninic acid (BCA) assays, are commonly used to quantify protein concentration. The accuracy of these assays depends on the amino acid composition. Because of the extensive use of reductive methylation in the study of proteins and the importance of biological methylation, it is necessary to evaluate the impact of lysyl methylation on the Bradford and BCA assays. Unmodified and reductively methylated proteins were analyzed using the absorbance at 280 nm to standardize the concentrations. Using model compounds, we demonstrate that the dimethylation of lysyl ε-amines does not affect the proteins' molar extinction coefficients at 280 nm. For the Bradford assay, the responses (absorbance per unit concentration) of the unmodified and reductively methylated proteins were similar, with a slight decrease in the response upon methylation. For the BCA assay, the responses of the reductively methylated proteins were consistently higher, overestimating the concentrations of the methylated proteins. The enhanced color formation in the BCA assay may be due to the lower acid dissociation constants of the lysyl ε-dimethylamines compared with the unmodified ε-amine, favoring Cu(II) binding in biuret-like complexes. The implications for the analysis of biologically methylated samples are discussed.     

Aqueous zymography screening of matrix metalloproteinase activity and inhibition based on colorimetric gold nanoparticles

An optical gold nanoparticles (AuNPs)-based method was fabricated for the rapid detection of matrix metalloproteinase (MMP) activity and screening potential MMP inhibitors without sophisticated instruments. The diagnosis platform was composed of AuNPs, particular MMP substrates and 6-mercapto-1-hexanol (MCH). The functionalized AuNPs were subjected to specific MMP digestion, and the MMP found the substrate on AuNPs, such that the AuNPs lost shelter and MCH increased the attraction force between AuNPs. Consequently, AuNPs aggregation and a color change from red to purple with increasing MMP concentration were observed. The surface plasmon resonance (SPR) of the formed AuNPs allowed for the quantitative detection of MMP activity. A sensitive linear correlation existed between the absorbance and the activity of the MMPs, which ranged from 10 ng/mL to 700 ng/mL in NTTC buffer and plasma samples. The proposed colorimetric method could be accomplished in a homogeneous solution with one-step operation in 30 min and has been successfully applied to the determination of particular MMP activity in plasma samples, in which the results are consistent with substrate zymography. This technology may become a simple platform for parallel screening a number of inhibitors and offer an alternative method to studying the efficiency of inhibitors for suppressing MMP activity. The absorbance ratio at 625 nm and 525 nm (A(625)/A(525)) confirmed the efficiency of the inhibitors as observed in substrate zymography. The IC(50) of ONO-4817 and galardin for MMP-1, MMP-2 and MMP-7 determined by the proposed colorimetric method was similar to the results of substrate zymography.     

New colorimetric method for the quantitative estimation of phospholipids without acid digestion

A unique colorimetric method for the quantitative determination of phospholipids that does not involve the acid digestion of the lipid is described. The phospholipids, after separation by thin-layer chromatography and elution from the silica gel, are heated with a chromogenic solution that is a modification of a spray reagent formulated by Vaskovsky and Kostetsky (1968, J. Lipid Res., 9: 396). The absorbance of the colored complex was read at 710 nm, and it followed Beer's law in the range of 1-10 micro g of phospholipid phosphorus.     

The reaction of nitric oxide with copper proteins and the photodissociation of copper-NO complexes

The reactivity with nitric oxide was investigated for a number of type-1, type-2 and type-3 copper proteins azurin from Pseudomonas aeruginosa (type-1 copper); bovine superoxide dismutase, diamine oxidase from pig kidney and galactose oxidase from Dactylium dendroides (type-2 copper); haemocyanin from Helix pomatia (type-3 copper); the blue oxidases ceruloplasmin from pig serum, and ascorbate oxidase from Cucurbita pepo medullosa. Type-1 copper formed complexes with NO in the oxidised state, which complexes were only fully formed at low temperatures and could be photodissociated at 77K. Complex formation led to the disappearance of the EPR signal of type-1 copper and of the optical absorbance band in the 600 nm region. In azurin, photodissociation caused the reappearance of the original 625 nm absorbance band, but in the blue oxidases, a new band with lower intensity was found at 595 nm instead of the original absorbance band at 610 nm. In all cases, the EPR signal of type-1 copper did not return. These results are best explained by the formation of a photolabile type-1 Cu1+-NO+ complex. They also indicate that in the complex formed, the type-1 copper structure is probably not disrupted, and that after illumination, the nitric oxide molecule is still in the near vicinity of the copper atom. Type-2 copper did not react at all with nitric oxide, and type-3 copper formed complexes with nitric oxide in both the oxidised and the reduced state, but photodissociation of these complexes could not be demonstrated.     

A Method for the Quantitative Assessment of Wound-induced Chitinase Activity in Potato Tubers

A method for the quantitative assessment of chitinase activity was developed. Dilution series of crude potato tuber chitinase extracts were assayed with a colorimetric microtitre plate assay. using CM-chitin-RBV as enzyme substrate. Linearity between absorbance values mea-sured (540 nm) and enzyme concentration was found to be limited to the low concentration range. where depletion of the substrate was no longer limiting. As as absorbance of 0.1 always fell within the concentration range for which absorbance-concentration linearity was valid. one unit of enzyme activity was defined as the amount of enzyme needed to yield and A of 0.1. A more reliable method for the assessment of chitinase activity was established by basing the difinition of enzyme, activity on a concentration rather than on as absorbance value. as was done previously. Using this method, differences in the rate of chitinase induction upon wounding were demonstrated for six commercial potato cultivars.