A diaphragm diffusion cell applied to ethanol diffusion in agarose gel: A reproducibility study

Summary The effective diffusion coefficient of ethanol in a 4 %(w/v) agarose gel at 25°C was measured using a diaphragm diffusion cell. The reproducibility was very good; a standard deviation of only 2% was obtained. The mean value (9.5×10⁻⁶ cm²/s) agreed well with available theory and previous investigations.     

A convenient automated method for the determination of proteolytic enzymes

An automated method for the assay of proteolytic enzymes is described. The insoluble dye-protein complex, Remazolbrilliant Blue-hide powder, is used as the substrate and is readily digested by trypsin, elastase, subtilisin, thermolysin, chymotrypsin, and to a lesser extent pronase, pepsin, and bacterial collagenase. The proteolytic activity of crude microbial culture preparations is expressed in terms of an equivalent concentration of crystalline trypsin, which itself can be readily determined within the concentration range 0.25–5.0 μg/ml.     

Radial diffusion in gel for micro determination of enzymes. I. Muramidase, alpha-amylase, DNase 1, RNase A, acid phosphatase, and alkaline phosphatase

The principle of radial diffusion in substrate containing agar gel has been applied for the quantitative assessment of several enzymes. Muramidase, alpha-amylase, DNase I, RNase A, acid phosphatase, and alkaline phosphatase have been investigated. Clearing zones in the opalescent agar, staining of the substrate incorporated in the agar, or a colored insoluble hydrolysis product indicate the diffusion zone of the enzyme. A linear relationship was found between the logarithm of the enzyme concentration and the corresponding diameters of the diffusion zones over a wide range. Standard dilutions of the different enzymes are used as reference.     

Peptide bond cleavage site determination of novel proteolytic enzymes found in ROS 17/2.8 cell lysates

We have identified proteolytic activities in the rat osteoblastic osteosarcoma cell line ROS 17/2.8 which are capable of cleaving a peptide substrate for protein kinase C-mediated phosphorylation (PSPKC, Pro-Leu-Ser-Arg-Thr-Leu-Ser-Val-Ala-Ala-Lys). Using polyacrylamide gel electrophoresis conditions similar to those used to resolve small molecular weight proteins, the peptide bonds of PSPKC which are cleaved by the proteolytic activities present in ROS 17/2.8 cell lysates have been determined. These activities cleave the Ser-Arg, Thr-Leu, and Ser-Val peptide bonds. To date, no proteolytic activities present in osteoblast cell lysates have been described with the aforementioned peptide bond specificities, suggesting that these activities are novel. The PSPKC-cleaved peptide fragment pattern generated was similar for several different osteoblast cell lysates. Lysates generated from different rat tissues were also able to cleave PSPKC, but the peptide fragment pattern generated by ROS 17/2.8 cell lysates appeared to be unique amongst these tissues. © 1996 Wiley-Liss, Inc.     

Improvements of the Anson assay for measuring proteolytic activities in acidic pH range.

This paper proposes a modification of the classic Anson assay [Anson, M. L. (1939) J. Gen. Physiol.22, 79–89] for proteolytic enzymes which are active in the acidic pH range; it abolishes many of the constraints common to this widely used method. The present procedure allows for: (i) the control of zymogen activation; (ii) the measurement of the hydrolyzed products on a very wide concentration range, due to their extended direct proportionality to both the incubation time and the enzyme concentration; (iii) the possibility to perform the assay at any given pH between 1.3 and 5.0 with minimal interference from ionic strength; (iv) a very simple conversion of the absorbance into proteolytic units, allowed by a better choice of TCA concentration. The method offers good sensitivity and is compared in detail to the original method and its various modifications.     

A rapid method for the determination of proteolytic activities of enzyme preparations

A method has been described for the determination of proteolytic activities of enzyme preparations using casein as substrate. The rate of digestion is proportional to the enzyme concentration used. This relationship is utilized as a measure of the enzyme activity. One unit of activity is defined as the amount which is required to digest casein in 15 minutes at 37.5 degrees C. so that 50 per cent of the protein in 1 ml. of 0.25 per cent solution is not precipitable by trichloroacetic acid. This method has been used to determine the activity of enzymes from different sources and also used to follow the rate of activation of enzymes.     

Incorporation of fluorescent enzyme substrates in agarose gel for in situ zymography

The currently available methods for the detection of proteases in tissue sections are characterized by limited substrate specificity and low sensitivity and are also cumbersome. We have developed a novel in situ zymography method that uses a synthetic substrate conjugated to a fluorescent tag for detection of proteases in tissue sections. In the presence of active enzyme, the fluorescent tag is cleaved off from the substrate peptide chain resulting in an approximately 100-fold increase in the fluorescent signal. In order to minimize the diffusion of the fluorescent tag, the substrate is incorporated into 1% agarose prior to overlaying onto the tissue section. This method retains the morphological details of the tissue section, is highly sensitive and specific for the designated peptide sequence, and provides information regarding the functional status of the enzyme. Thus, this method could be used for detection and monitoring of enzymatic activity in tissue sections for a variety of applications.     

Proton-dependent dissociation equilibrium of hemoglobin. 1. A 700-nanometer light-scattering study on horse methemoglobin in the pH range 4.8 to 7.2.

The effect of proton concentration upon the subunit dissociation of horse methemoglobin has been investigated at two ionic strengths by light scattering photometry at 700 nm. Differential refractometry revealed a slight but systematic decrease of the specific refractive index increment with decreasing protein concentration for solutions in dialytic equilibrium with the solvent. In the pH range 4.8-7.2 the dissociation can be described by a simple equilibrium between tetramers and dimers. The dissociation constant Kd of the met derivative is found to be very similar to those of the O2- and CO-ligated states. From the slope of a plot of log Kd vs. pH, the number of protons bound is n = 1.3 +/- 0.1 resulting from an increase in the pK values of two groups upon dissociation. These two groups must be identical because the dissociation is symmetrical.     

Measurement of the diffusion coefficient of ethanol in agarose gel beads: A reproducibility study

Summary The diffusion coefficient of ethanol in a 4 w/w% agarose gel at 25°C was measured, using the methods of unsteady-state diffusion into, and out of, gel beads dispersed in a solution of finite volume. The results (8.0 – 9.5×10^–6 cm²/s) agreed well with available theory. The results of method out were more reproducible than method in, but the standard deviation was in no case lower than 3.3%.     

A study of the apolar interaction of the enzyme rhodanese with octyl substituted agarose gel

The accessibilities of sites on the surface of the enzyme rhodanese for binding to macromolecular apolarity have been measured for the two forms of the enzyme related to obligatory catalytic intermediates: the free enzyme, E and the sulfur substituted enzyme, ES. This study was done using a micromethod developed for this purpose which allows facile assessment of the apolar binding of proteins to commercially available beads of cross-linked agarose on which hydrophobic groups have been immobilized. The results indicate that the enzyme rhodanese can bind to macromolecular apolarity and that there is considerably more binding of the E form than the ES form. The fact that the binding is relatively slow implicates a protein conformational change in the rate limiting binding step. In fact, there is a large increase in the binding when the temperature is raised from 23° to 40° which correlates with previous results showing a conformational change in rhodanese over the same temperature range. These results in comparison with other solution studies and with x-ray studies are consistent with a model for rhodanese which has an apolar active site and a mechanism for catalysis that includes a conformational change.     

Screening for bacilli producing cellulolytic enzymes active in the neutral pH range

S. LANDAUD, P. PIQUEREL AND J. POURQUIÉ. 1995. More than 50 soil samples of various geographical origins have been screened for aerobic sporeformer bacteria displaying cellulolytic activity in the neutral pH range. Six independent clones were selected, which utilize and hydrolyse amorphous cellulose when grown at pH 8 on agarsolidified media. These clones belong to different species of the Bacillus genus. Their hydrolytic potential is not restricted to cellulose but extends to a range of polysaccharides. When grown in liquid cultures, two clones were shown to produce extracellular cellulolytic enzymes active in the pH 6–8 range.