Viscometric assays

A viscometric assay and identification of hydrolysis products were used to determine the mechanism of action of PG. An endo-PG is characterized by a strong reduction in viscosity (e.g. 50%) with a concomitantly low (e.g. 1-3%) release of reducing groups [9]. The time required for 50% decrease in viscosity of a 3.0% (w/v) sodium polypectate solution at 25°C was approximately 10 min, at which time about 1.5% of the total galacturonide bonds had been hydrolysed (data not shown). These results reveal a random mechanism of hydrolysis of sodium polypectate and the enzyme was a poly oc(l,4)-D-galacturonide glycanohydrolase (EC 3.2.1.15) or endo-PG. 

The reaction conditions chosen for the assays are based on published optimal conditions for PGase enzymes. These enzymes typically have maximal activities at slightly acidic pH (Tucker and Seymour, 2002) and, in general, appear to be relatively stable at temperatures from 30° to 40°C. Optimal reaction conditions are likely to be enzyme specific, so one may have to alter the conditions to match the properties of the enzyme of interest. In all cases, the analyst should take into account the properties of the substrate, particularly its solubility, as well as the properties of the enzyme. For example, because solutions of polygalacturonic acid tend to gel as the pH is lowered below 3, viscometric assays (Basic Protocol 2) at these relatively low pHs are often not feasible. 

Manning, K., Improved viscometric assay for cellulase methods. J. Biochem. Biotechnol. 1981, 5, 189-202. 

The Commission on Enzymes states that, wherever possible, enzyme assays should be based on measurements of initial rates of reaction. A viscometric assay method should be especially suitable for this purpose since the mean molecular weight of a polymer can be rapidly determined by such methods. A useful viscometric method has been worked out by Almin et al. (3,4, 5). 

The stability of pectate and pectin depolymerizing enzymes produced by Mucor puriformis, Rhizopus sexualis, R. stolonifer, Botrytis cinerea, Aureobasidium pullulans, Trichosporon pullulans, and Cryptococcus albidus var. albidus in sulphite liquor has been studied in relation to the breakdown of sulphited strawberries. Marked breakdown of fruit occurred only when pectolytic activity could be detected in the liquor for more than two weeks using a viscometric assay. Of the fungi tested, Rhizopus species produced enzymes that were the most stable in sulphite liquor. For each of the Mucor and Rhizopus species tested, the stability of poly-D-galacturonases in sulphite liquor was very similar for extracts of infected fruit and culture filtrates. It was suggested that sulphite labile (=acid labile) and sulphite stable (=acid stable) forms of the poly-D-galacturonases are present. 

The mucin clot prevention test, the stringiness test, the AGRA test, and the viscometric assay measure changes in the high polymeric properties of hyaluronic acid. The turbidimetric method determines the amount of unreacted substrate, and the reductometric method estimates the number of glucosidic linkages which have been hydrolyzed. 

Humphrey and Jaques (80) assayed several different enzyme preparations by the turbidimetric method (190), the viscometric method (106), and an improved biological method based on skin diffusion (83). The two in vitro methods were carried out in identical buffers at pH 7 in order to approach physiological conditions. The reaction time for the turbidimetric assay was 10 minutes and for the viscometric assay, 20 minutes. All activities were expressed in terms of a reference standard for hyaluronidase prepared from testicular material. Two substrates of different purity were employed. The correlations were generally better with the more purified hyaluronate. Testicular hyaluronidases showed very satisfactory agreement between in vitro and in vivo assays. Enzyme samples from staphylococci and streptococci showed poorer correlation. This may be attributed to the heterogeneity of these preparations. 

A viscometric assay for the alginolytic activities in bacteria has been reported. ... 

This is a very useful assay because it provides a means to quantitate the action of collagenase on collagen fibrils. The method contrasts with the viscometric assay which uses collagen in soluble form. The method was developed by Nagai et al. (1966), and modified slightly by Lazarus et degradation products from... 

Gusakov, A.V., Markov, A.V., Grishutin, S.G., Semenova, M.V., Kondratyeva, E.G., and Sinitsyn, A.P. 2002. Viscometric method for assaying of total endodepolymerase activity of pectinases. Biochemistry (Moscow) 67 676-682. 

The methods described below outline three dynamic adhesion/aggregation assays used to assess the in vitro and/or ex vivo efficacy of platelet antagonists (1) a viscometric-flow cytometric assay to measure shear-induced platelet-platelet aggregation in the bulk phase, (2) a perfusion chamber coupled with a computerized videomicroscopy system to visualize in real time and quantify (a) the adhesion and subsequent aggregation of platelets flowing over an immobilized substrate (e.g. extracellular matrix protein) and (b) free-flowing monocytic cell adhesion to immobilized platelets. 

A third technique for determination of alpha-amylase is the viscometric method. This is the most sensitive method for studying the initial attack on amylose, but it is too time-consuming to be used as a routine method for enzymic assay. If the relation between viscosity and molecular weight is known for the substrate, an estimate of the rate of bond scission may be made. (This estimation would be valid only if completely random attack were occurring.)... 

In Reference 8 it is mentioned that the enzyme yield is low upon fractionation on Sephadex columns. We have found the yield on both Sephadex gels and polyacrylamide gels to be low when the cellulases from C. lignorum are fractionated. In a system of ammonium acetate buffer pH 5.0, 0.1M the enzyme yield was 31% on a Sephadex column and 37% on a polyacrylamide gel column. Evidence exists that the yield is dependent upon the buffer system and the pH value of the buffer system. Different buffer systems for gel filtration are now studied in our laboratory in order to find out if the yield can be increased. One of the reasons why the low yield of cellulases upon gel filtration has not been noticed before is that the activity of these enzymes has usually been assayed viscometrically using only relative enzyme units as a basis. 

The spot test described herein is based on the Viscometric Affinity Assay (VAA) described earlier [/, 2]. The principle of the VAA is based on the fact that the mixture of the glucose-specific lectin Concanavalin A (ConA, [3,4]) with a highly concentrated dispersion of 1,000 kDa dextran (wt 1-10%) yields a tremendous rise of the resulting viscosity (up to 20 times). Such viscous dispersions can adopt gel-like properties as discussed in detail by Ehwald et al. [5]. This viscous behavior is due to extensive intermolecular affinity crosslinking of dextran molecules by ConA (see Figure la). 

Figure 1. Schematics of mechanism of the Viscometric Affinity Assay (VAA).

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