A simple assay with dyed substrates to quantify cellulase and hemicellulase activity of fungi

Summary A simple assay is described for the quantitative measurement of cellulase and hemicellulase activities. It is based on the enzymatic release of stained cleavage products originating from dyed substrates such as different celluloses, mannan and xylan, diffusing from an upper into a lower agar layer. The amount of cleavage products is assessed by direct absorbance measurement in the agar tubes employing an ordinary filter photometer. The method proved to be applicable to various species of basidiomycete fungi.     

Standardized Viral Hemagglutination and Hemagglutination-Inhibition Tests. I. Standardization of Erythrocyte Suspensions

A spectrophotometric procedure for standardizing red blood cell suspensions for use in viral hemagglutination tests is described. The procedure is based on highly reproducible cyanmethemoglobin absorbance readings at 540 nm on any suitable spectrophotometer. Target values for milligrams of cyanmethemoglobin per 100 ml are given for the red cell suspensions of all mammalian and avian species employed in viral hemagglutination tests. By use of these values and a cyanmethemoglobin standard curve for a particular photometer, approximate 4% erythrocyte suspensions can be diluted to any lesser concentration. Coefficients of variation for the various diluted suspensions range from 4 to 7%.     

Double-beam flying spot scanner for two-dimensional polyacrylamide gel electrophoresis

The construction of a double-beam photometer in which the light source is a cathode ray oscilloscope is described. The light spot from the oscilloscope was focused and reduced in size at the gel plane to give a diameter of less than 0.15 mm and make it possible to scan over a 50 X 59-mm rectangle; using reduced spatial resolution (spot less than 0.2 mm) the area scanned becomes 70 X 90 mm. The light from the CRT was divided into two beams; one was directed through the transparent object to a photomultiplier and the other to a reference photomultiplier. The signals from these two detectors were converted to the logarithm of the ratio by a logging amplifier to give a direct measure of absorbance. Positioning of the spot, control of light intensity, and measurement of absorbance were carried out through an interface to a 16-bit computer. The relationship between measured and actual absorbance was linear over the range of absorbance 0 to 2, which could be raised to 1 to 3 by placing a neutral filter in the reference beam. The system generated an image containing 256 X 256 pixels in about 5 min, the scanning speed was determined by the persistence time of the P4 phosphor on the cathode ray tube, and faster scans can be made using A6 phosphor.     

A simplified method for screening acetylcholinesterase inhibitors by the flesh fly (Dipt., Sarcophagidae) cell culture system

A simplified economical method for assaying acetylcholinesterase inhibitors was devised. A flesh fly, Sarcophaga peregrina Robineau-Desvoidy, cell line, NIH-SaPe-4, was cultured in 96-well microplates at 25°C. After 2 days culture, the culture media was removed and phosphate buffer was introduced. The cells were disintegrated by freezing–thawing, and a reaction mixture containing substrate, inhibitor, and colour developer was added. The change of absorbance at 405 nm at 25°C was measured with a microplate photometer and the concentration of the test substance required to inhibit the enzyme reaction by 50% (I₅₀) was calculated.     

An automated continuous determination of oxygen with high sensitivity

A sensitive, rapid, and precise method is described for the continuous determination of oxygen in gases. The principle of the method is based on the reaction of O₂ with an alkaline catechol or pyrogallol solution, which is combined with a Fe²⁺ solution to increase the sensitivity of the color reaction. The development of the color takes place in a tube system provided with a proportioning pump and is read automatically on a recorder after passing a flow cell of a photometer. The lower limit of sensitivity of this method is 0.05 μl of O₂ min^?1. Thus, in a gas flow of ≈0.5 ml min^?1, an oxygen concentration of ≥0.01% (v/v) can be determined. If the gas flow is increased to ≈2.5 ml min^?1, this limit of sensitivity is lowered to ≥0.002% (v/v). Since a 2-min period is necessary for the measurement, the volume of the sample has to be 1 ml in the first case and 5 ml in the second.     

Cytophotometry of post mortem glycogenolysis in quiescent bovine muscle fibres in relation to temperature, succinate dehydrogenase activity and adenosine triphosphatase activity

Summary Immediatepost mortem samples of sternomandibularis muscles from six steers were maintained with a minimum of intrinsic activity at approximately 40°C or allowed to cool to approximately 22°C. Samples were frozen in liquid nitrogen at 0, 2, 4 and 6hpost mortem and serial transverse sections were stained by the periodic acid-Schiff (PAS) reaction for glycogen or reacted for adenosine triphosphatase (ATPase) or succinate dehydrogenase. Individual fibres were mapped and categorized from their ATPase and succinate dehydrogenase activity using a projecting microscope. The absorbance of PAS-stained glycogen in individual fibres was measured with a microscope photometer at 570 and 601 nm with a correction for distributional error. Overall, thepost mortem decline in absorbance was approximately twice as fast in body temperature samples relative to room temperature samples. Transientpost mortem increases in absorbance were detected in some situations, particularly in fibres with strong ATPase activity from room temperature samples. In fibres with strong ATPase activity, the rate of decline in absorbance increased progressivelypost mortem. Fibres with weak ATPase mostly had a lower initialpost mortem absorbance and were generally the first to become PAS-negative.     

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.     

The influence of photometer design on optical-conformational changes.

When cells and large subcellular structures suffer a change in volume or internal structure, their light-scattering properties are normally altered. These optical-conformation changes are potential sources of information about conformation and processes which alter it. Classical light-scattering theory for spherical particles is used to determine how the transmittance or extinction of a cell suspension should respond when such a conformational change occurs and the measurements are made with a conventional photometer. This extends an earlier study of transmittances measured with an “ideal” photometer. The photocell of an ideal instrument collects only the directly transmitted light. In a conventional instrument it also collects the light scattered at small angles, which is usually most of the scattered light. Extinction (optical density, absorbance) of suspensions of spherical cells was computed for several photometer designs. It is found that γ, the angle of acceptance of the photocell, has a significant influence on the extent and even the nature of the photometric response to a given conformational change. Earlier, it was shown that a decrease in cell volume or increase in internal structure will increase extinction for cells of many sizes. Now it is found that a large γ-value increases these effects. An approach to the interpretation of transmitted light fluxes in terms of theoretical predictions is outlined.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.     

A spectrophotometric method for the determination of the catecholase activity of tyrosinase by Besthorn's hydrazone

A quick and easily reproducible procedure for determining the catecholase activity of tyrosinase is described. The o-benzoquinone formed during the oxidation of catechol is condensed with Besthorn's hydrazone. The absorbance of the condensation compound, extracted with chloroform, is read at 500 nm.     

Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein

A method has been developed to determine the relative or actual number of attached cells in microtiter plate wells without making direct cell counts. The procedure is based upon staining total cellular protein with Coomassie brilliant blue G-250, followed by measurement of absorbance at 630 nm in a spectrophotometer designed to read each well of a 96-well microtiter plate. No destaining of cells is required. A linear correlation exists (r = 0.970) between cell number and absorbance over a useful range. Intraplate well-to-well variation is acceptable (CV = 0.101). This method was used to measure the proliferative response of human vascular smooth muscle cells to human serum. It should be useful in other assays involving proliferation of attached cultured cells.     

Adaptation of E. coli cell method for micro-scale nitrate measurement with the Griess reaction in culture media

The E. coli cell method for nitrate measurement consists of two-steps: nitrate reduction by the E. coli cell usually under anaerobic conditions and subsequently nitrite measurement with the Griess reaction. It was found that the E. coli DSM 498k wildtype cell can reduce nitrate to nitrite under aerobic conditions. Therefore, the E. coli method for nitrate measurement was adapted to be performed under aerobic conditions in a microtiter plate. The adapted method is simpler than the original E. coli method and other nitrate methods such as those with inorganic reductants and with purified enzymes. Furthermore, it was found that for the Griess reaction the pH values of samples after addition of the Griess reagent A should be lower than 1.8 for a stable absorbance at 540 nm to be reached. It is important to add the two Griess reagents separately and to read the absorbance twice consecutively in a microtiter plate. The adapted E. coli method was successfully applied to measure the traces of nitrate in MRS and other medium components by measuring the standard curve of a dilution of each individual medium component. It was found that many organic medium components contain traces of nitrate, while none of them contain detectable nitrite. Among these, the extract of meat and yeast extract contain relatively high amounts of nitrate: 217 mg N/kg and 99 mg N/kg respectively. MRS broth contains nitrate from 0.3 to 0.6 mg N/l depending on the batch numbers of the product. The adapted E. coli can also be used for nitrate measurement in other matrices.     

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.     

Studies on lsoamylase

A method for determining the isoamylase activity is devised. A properly diluted enzyme solution is allowed to act at 20°C on a 1% solution of glutinous-rice starch buffered to pH 6.2. After 24 hours, 0.2 cc of the reaction mixture is mixed with 2 cc of 0.01 N I₂ solution, and diluted to 25 cc. Optical density (E) of the solution at 620 mµ is read in a Pulfrich photometer, using a 1-cm cell. The increment of E at 620 mµ is proportional to enzyme activity within certain ranges. The amount of enzyme is expressed in the “isoamylase unit”, i.e., an increase of E 0.200 being taken as 10 units.

A pH-activity curve is given for yeast isoamylase, which shows a sharp maximum at pH 6.2.     

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.     

Characterization of the unfolding of ribonuclease A in aqueous methanol solvents

The effect of methanol on the thermal denaturation of ribonuclease A has been investigated over the -40 to 70 degrees C range. The transition was fully reversible to at least 60% (v/v) methanol at an apparent pH of cryosolvent (pH) of 3.0 and was examined at methanol concentrations as high as 80%. The unfolding transition, as monitored by absorbance change at 286 nm, became progressively broader and occurred at increasingly lower temperatures as the alcohol concentration increased. In 50% methanol, increasing the pH from 2 to 6 shifted the transition to higher temperature. A substantial decrease in cooperativity was noted at the more acidic conditions. On the other hand, increasing concentrations of guanidine hydrochloride in 50% methanol caused the transition to shift to lower temperatures with little effect on the cooperativity. The observed effects on the cooperativity of the unfolding transition suggest that methanol and lower temperatures may increase the concentration of partially folded intermediate states in the unfolding of ribonuclease. Comparison of the transition in 50% methanol as determined by absorbance or fluorescence, which monitor the degree of exposure of buried tyrosines and hence the tertiary structure, to that determined by far-UV circular dichroism, which monitors secondary structure, indicated that the major unfolding transition occurred at a higher temperature in the latter case. Thus, the tertiary structure is lost at a lower temperature than the secondary structure. This observation is consistent with a model of protein folding in which initially formed regions of secondary structure pack together, predominantly by hydrophobic interactions, to give the tertiary structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of flattening coefficient for absorption and circular dichroism using simulation

The absorbance and circular dichroism (CD) of suspensions is lower than if the same amount of chromophore were uniformly distributed throughout the medium. Several mathematical treatments of this absorption flattening phenomenon have been presented using various assumptions and approximations. This article demonstrates an alternative simulation approach that allows relaxation of assumptions. On current desktop computers, the algorithm runs quickly with enough particles and light paths considered to get answers that are usually accurate to better than 3%. Results from the simulation agree with the most popular analytical model for 0.01 volume fraction of particles, showing that the extent of flattening depends mainly on the absorbance through a particle diameter. Unlike previous models, the simulation can show that flattening is significantly lower when volume fraction increases to 0.1 but is higher when the particles have a size distribution. The simulation can predict the slope of the nearly linear relationship between flattening of CD and the absorbance of the suspension. This provides a method to correct experimental CD data where volume fraction and particle size are known.     

Cytophotometry of post mortem glycogenolysis in different histochemical types of muscle fibres of the pig.

Synopsis Serial transverse sections of porcine longissimus dorsi muscle (18 pigs, 50 to 178 kg live weight) were reacted for NAD tetrazolium reductase and ATPase at pH9.4, and for glycogen with the periodic acid-Schiff (PAS) reaction. Three histochemical types of muscle fibre were identified; (1) strong ATPase and weak NADH oxidative activity; (2) strong ATPase and intermediate NADH oxidative activity; and (3) weak ATPase and strong NADH oxidative activity. Immediatepost mortem samples from one side of each animal were compared with a laterpost mortem sample from the other side by measuring the absorbance of PAS-stained glycogen at 570 nm with a microscope photometer. Laterpost mortem absorbance was expressed as a percentage of immediatepost mortem absorbance in each category of muscle fibre in order to compensate for distributional error and different starting levels of glycogen. Muscle fibres with weak ATPase and strong NADH oxidative activity showed a progressive decrease in absorbance of PAS-stained glycogenpost mortem. In some animals, fibres with strong ATPase and intermediate or weak NADH oxidative activity showed an initialpost mortem increase in absorbance of PAS-stained glycogen which was then followed by a progressive decrease. The maximum rates of decrease in absorbance in the three fibre types did not differ to any great extent.     

Enzyme-linked immunosorbent assay for human plasma apolipoprotein B

A noncompetitive enzyme-linked immunosorbent assay (ELISA) has been developed for measuring total plasma apolipoprotein (apo) B using affinity purified polyclonal and monoclonal antibodies. Microtiter plates from different manufacturers were tested with regard to their IgG binding characteristics; only one plate yielded consistent coefficients of variation of less than 5%. The optimal plasma dilution in this assay was 1:3000. IgG anti-apoB antisera conjugated to alkaline phosphatase was used as a second antibody. p-Nitrophenyl phosphate was utilized as substrate for color development, and the absorbance (410 nm) was read utilizing an ELISA reader interfaced with a microcomputer for data processing. Plasma apoB levels in plasma have been determined in 1115 male and female participants in the Framingham Offspring Study. Mean (+/- SD) plasma concentrations were 89 +/- 28 mg/dl. Significant age and sex related differences in apoB levels were noted.     

Investigation of granulocytopoietic kinetics by microdensitometric evaluation of primary granule naphthol-AS-D-chloroacetate esterase activity

Using a scanning microscope photometer we determined quantitatively the enzymecytochemical reaction product for naphthol-AS-D-chloroacetate esterase in neutrophilic granulocytes and their precursors in man. Evaluation of neutrophilic cells from three healthy donors resulted in a logarithm-normal distribution. After subdivision of these cells in their morphologically defined maturational stages no statistically bimodal distribution was shown within the single cell groups. Myelocytes showed twice the amount of the polymorphonuclear neutrophil absorption values. The highest promyelocyte obsorptions were double the values of the myelocyte absorptions. The standard deviation of the absorbance obtained with promyelocytes (which encompass cells already producing granules up to cells reaching their maximal granule content) was significantly higher than the standard deviation of the myelocytes. As already known, primary granules are only synthesized at the promyelocyte stage and - according to the present knowledge - their chloracylesterase and peroxidase activities are not lost during further maturation. Consequently, our results indicate that only enzyme-rich, late promyelocytes undergo mitosis transforming into myelocytes. Correspondingly, their absorption value was halved. Since the absorbance from myelocytes to polymorphonuclears is again halved, myelocytes divide only once. Metamyelocyte absorptions in part correspond to that of myelocytes. This indicates that no distinction can be made between myelocytes with mitotic capacity and "true" if only the size and the nuclear shape are considered metamyelocytes which are not longer capable of undergoing mitosis.