A colorimetric method for measuring the esterolytic activity of elastase

This paper describes a new colorimetric method for measuring the activity of elastase. The substrate used is acetyl-l-alanyl-l-alanyl-l-alanine-methyl ester. Its concentration is determined before and after enzymatic action by the hydroxamic acid method of Hestrin. The procedure described is simpler and more rapid than the usual potentiometric method and may be adapted to serial assays. With a 1-hr test, elastase concentrations as low as 0.2 μg/ml may be assessed accurately.     

Bead method for measuring the root surface area in conifers

A method of determining the surface area of mycorrhizous conifer roots is described. The roots are cleaned from soil particles by washing, slightly dried and covered with a monolayer of plastic beads. The surface area is estimated on the basis of the weight of adhered beads.The method has been tested against the surface area measurements by a photographic-planimetric method. The estimates of the surface area by both methods compare fairly well.An analysis of model root systems shows that the accuracy of the method for measuring the roots of 0.3mm diameter is about 5%.     

A colorimetric procedure for measuring beta-lactamase activity

The enzymatic hydrolysis of benzylpenicillin was measured by a novel colorimetric procedure. The penicilloic acid generated from the hydrolysis of penicillin was reacted with CuSO4 and neocuproine to form a colored complex having a maximal absorption at 454.5 nm. A plot of absorbance versus beta-lactamase activity yielded a straight line from 1 to 5 mU of enzyme. Using TEM-1 as the model beta-lactamase, a Km of 46 microM was observed with benzylpenicillin serving as the substrate. When the assay was used to determine levels of benzylpenicillin, the absorbance was found to be linearly proportional to exogenously added penicillin from 2.8 to 88 microM. This procedure is simple to use and can be employed to measure the hydrolysis of other beta-lactam antibiotics.     

A method for determining the surface area of corals

Although there are several techniques available that can accurately determine the surface area of simple branching corals, there is an absence of techniques that can be applied to finely branching species like Pocillopora damicornis. This paper describes a rapid, spectrophotometer-based technique that can accurately determine the surface area of a range of coral species, including P. damicornis. The technique involves dipping corals coated with plastic varnish into a solution that contains a small amount of detergent and the dye Methylene Blue. The amount of dye solution clinging to the surfaces (as a thinlayer) is proportional to the total surface area.     

A sensitivity analysis of the 'Transmittance-Reflectance' method for measuring light absorption by aquatic particles

A thorough sensitivity analysis of the ‘Transmittance–Reflectance’(T-R) method for measuringin vivo light absorption by aquaticparticles retained on glass-fiber filters has been carried out.Particular attention has been devoted to the error contributionby the variability of the optical properties of the GF/F filtersused. The overall error of the measurement of the filter-retainedparticle optical density, OD_s, has been evaluated as ~0.002(1), with 0.0015 error contribution by the filter variability.The corresponding error of the optical density of the particlesuspension, OD_sus, has been obtained by differentiation of theequation expressing the experimental correlation (OD_susversusOD_s); the error increases with the optical density value, from= 0.0015 (for OD_sus = 0.05) to = 0.027 at the upper limit ofthe optical density range tested (OD_sus = 0.59). The validityrange of the experimental (OD_sus versus OD_s) correlation hasbeen shown to extend to OD_s = 0.75. The importance of the accuratecorrection for light backscattering performed by the T-R methodhas been investigated by comparison with results given by thestandard ‘Transmittance’ (T) method; it has beenshown that the coarse correction for light scattering includedin the T method may cause serious errors in the measured opticaldensity spectra of mineral detritus. This evidence confirmsthe need to resort to the T-R method for the measurement ofparticle samples of detritus-rich coastal waters. The papersummarizes the latest developments of the T-R method and includesan Appendix with asynopsis of the routine now used by the authors.     

A quantitative colorimetric method of measuring alkaline phosphatase activity in eukaryotic cell membranes

Alkaline phosphatase (ALP) is glycoprotein structured metalophosphatase with several defined functions. It is present in many tissues of all living beings from bacteria to mammals. The enzyme may catalyse the hydrolysis of various monophosphate esters at alkaline pH. The objective of this study was to quantify ALP functioning particularly in the membranes of eukaryotic cells. The membranes of seven different cells (myeloma cells; hybrid cells; erythroleukaemia cells; lymphocytes and erythrocytes) were tested for ALP activity using a cellular enzyme assay, which is based on the conversion of para-nitrophenylphosphate (p-NPP) to para-nitrophenol and the colorimetric determination of the resulting coloured product. The test system was optimised with respect to substrate concentration, reaction time and the number of cells used as a source of enzyme. The obtained values were converted to quantitative results through a standard curve created using commercial ALP. In order to determine the effect of serum concentration on enzyme activity, 1G2 hybridoma, which is among the cells used in this study and which synthesizes monoclonal antibody against human serum albumin, was produced in different serum concentrations ranging from 0 to 15%.     

A C-assay for photorespiration in aquatic plants

The ¹⁴C-assay developed by Zelitch to evaluate photorespiration was modified for use in submersed aquatic plants in the laboratory and in situ. Results in laboratory cultures of Najas flexilis suggest that photorespiration occurs but is limited in comparison to terrestrial C₃ plants and is related to the rate of diffusion of CO₂ from the plant and to the carrying capacity of water for dissolved oxygen.     

A colorimetric assay for measuring the lysis of a plasma clot.

The present study describes a simple, quantitative assay for measuring the lysis of a plasma clot. The principle of the assay is based on the release of Coomassie brilliant blue R-250 dye from the clot. Thirty microliters of freshly prepared Coomassie brilliant blue R-250 (1 mg/ml) was added to 200 microliters of diluted human plasma (1:5). After mixing, 100 microliters of thrombin (2.5 NIH units/ml) were added to mediate a plasma clot. One milliliter of streptokinase (0.1 mg/ml) was used as a plasminogen activator to initiate clot lysis. During the course of lysis, 100 microliters of soluble material were transferred to microtiter wells and the absorbance at 540 nm was determined as a measure of clot lysis. This assay was used to measure clot lysis in 18 human plasma samples. The colorimetric method (X) developed in this report correlated well with that determined using a conventional 125I-fibrinogen method (Y): Y = 0.83X + 7.98 (r = 0.91).     

Allelopathic aquatic plants for aquatic weed management

This report presents, results of a feasibility study of use of allelopathic aquatic plants for aquatic weed management. In order to establish a list of potential allelopathic plants, we selected 16 aquatic plants native to the southeastern United States and subjected them to two bioassays — one involving lettuce seedlings and one involving the aquatic plantLemna minor as the target species. The lettuce seedling bioassay was selected because it is a widely used, experimentally simple assay to determine allelopathic activity. However, it uses lettuce, a terrestrial plant, as the target species, and thus may be less appropriate for use with aquatic plants. TheL. minor assay involves an aquatic plant as the target species and so is more appropriate for our goals, but it is experimentally much more complex and time-consuming. The plants selected for study wereBrasenia schreberi, Cabomba caroliniana, Ceratophyllum demersum, Eleocharis acicuiaris, Eleocharis obf usa, Hydrilla verticillata, Juncus repens, Limnobium spongia, Myriophyllum aquaticum, Myriophyllum spicatum, Najas guadalupensis, Nymphaea odorata, Nymphoides cordata, Potamogeton foliosus, Sparganium americanum, and Val/isneria americana.Nymphaea odorata (leaves and petioles) inhibited 78 % of lettuce seedling radicle growth and 98 % ofL. minor frond production. Brasenia schreberi inhibited 82 % of lettuce seedling radicle growth and 68 % of L. minor frond production. These results suggest thatN. odorata andB. schreberi are both highly inhibitory and are therefore candidates for use in aquatic weed management. Results also suggest that the simple lettuce seedling assay is a reasonable first “easy” one to use in an attempt to determine allelopathic potential of aquatic plants.