Proteolytic activity, assay

As an example, this protocol describes how to perform the reaction using a casein substrate and a commercial proteolytic enzyme cocktail. The activity of the commercial four-enzyme cocktail is 35 U/ml, but should be tested prior to the experiment in order to determine the appropriate amount of enzyme needed for the reaction. The proteolytic activity assay (Garcia-Carreno et al., 1994) is described below. 

Apart from the application of dyed proteins as substrates of proteolytic activity assay so far mentioned (see also 7.2), proteins are labeled with chromophores for studying membrane surfaces, for detecting proteins after several different [ ation procedures (i.e. chromatographic and electrophoretic techniques), for the determination of proteins in the mixture with many other substances, etc. 

The authors of this review have used I-human serum albumin for determining proteolytic activity in various types of foodstuffs, enzymatic preparations (determination of the contaminating proteolytic activity), pharmaceuticals and other materials Proteolytic activity assays using radioactively labeled substrates... 

The application of substrates labeled with various chromophores has become more and more popular in recent years. It has been already shown that proteolytic activity assays using dyed proteins have a long tradition (see 6.1). Their enforcement has not, however, been simple. There have been objections (often correct) against a discutable correlation between the colour intensity and the number of split peptide bonds. This disadvantage can be overcome by specific binding of a certain dye to the amino acid involved in the proteolytic breakdown of the protease being assayed. 

Proteolytic enzymes in the respiratory mucosa play important role(s) in the regulation of lung inflammation and remodelling [123, 124], Pulmonary proteolytic enzymes, however, also comprise one of the barriers which pulmonary-administered protein/peptide drugs have to overcome in order to achieve adequate bioavailability [125]. Intriguingly, the pulmonary enzymatic barrier is an aspect that has been little investigated and is poorly understood. Inconsistencies in the data available to date are most likely a result of the use of different techniques (e.g., PCR, immunotechniques and enzyme activity assays), different species and different cell (pheno)types, for example primary cells vs. cell lines. 

The methods used were acrosin proteolytic activity (APA) assay (with gelatin) and acrosin activity assay with N-a-benzoyl-DL-arginine-p-nitroanihde (BAPNA)-Triton X 100, and BAPNA assay for trypsin activity [9-11]. The antioxidant activity was tested spectrometrical with ABTS+ [12]. Cytotoxicity of extracts was determined by MTT Cell Proliferation Assay [13]. 

J.O. Capobianco, C.G. Lerner, and R.C. Goldman, Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase, Anal. Biochem., 204, 96, 1992. 

Proteolytic activities were assayed in 50 mM sodium acetate, pH 2.7, 1% casein or hemoglobin at 30°C. The activation of bovine trypsinogen was done with 5 mM trypsinogen in 50 mM sodium citrate, pH 3.0 at 30°C. Parentheses indicate the percentages of the activities of wild-type aspergillopepsin I. 

The stability of both the wild-type and mutant proteins is expressed as the melting temperature, Tm, which is the temperature at which 50% of the enzyme is denatures during irreversible heat denaturation. To prevent the self-digestion of penicillolysin, heat treatment was carried out at pH 5.0 because the proteolytic activity is substantially reduced at this pH and the active form of the enzyme is stable. For the wild-type enzyme and the three mutants, the thermal stabilities and the 7m changes were assayed at pH 5.0 by measuring the far-UV CD spectrum at 222 nm as a function of temperature. For the wild-type penicillolysin, the Tm... 

Fig. 12.1 Effect of temperature on the proteolytic activity of aqualysin I.15 The enzyme activity was assayed at the indicated temperatures in the presence ( ) and absence (O) of 1 mM CaCh.

Application and Principle This procedure is used to determine the proteolytic activity of papain, ficin, and bromelain. The assay is based on a 60-min proteolytic hydrolysis of a casein substrate at pH 6.0 and 40°. Unhydrolyzed substrate is precipitated with trichloroacetic acid and removed by filtration solubilized casein is then measured spectrophotometri-cally. 

Lipase (Microbial) Activity for Medium- and Long-Chain Fatty Acids, (S3)105 Lysozyme Activity, (S3)106 Maltogenic Amylase Activity, 804 Milk-Clotting Activity, 805 Pancreatin Activity, 805 Pepsin Activity, 807 Phospholipase A2 Activity, 808 Phytase Activity, 808 Plant Proteolytic Activity, 810 Proteolytic Activity, Bacterial (PC), 811 Proteolytic Activity, Fungal (HUT), 812 Proteolytic Activity, Fungal (SAP), 813 Pullulanase Activity, 814 Trypsin Activity, 814 Enzyme Assays, 786 Enzyme-Hydrolyzed (Source) Protein,... 

The first is the presence of proteolytic activities, which must be inhibited early in the procedure to prevent the degradation of other enzyme proteins. Second, the amount of protein present in these fluids is usually in excess of what an HPLC analytical column can handle without becoming clogged. And finally, these fluids often contain many low molecular weight compounds, either those added as nutrients or those present as a result of cellular metabolism. Since such compounds may resemble either the substrate or product, or both, of the enzymatic reaction under study, their presence in the reaction mixture could interfere with the assay. At the very least, such compounds will pass through the analytical column and appear on a chromatogram, confusing the experimental results. 

Low molecular weight compounds may be removed, and a variety of methods are available, including dialysis and gel filtration chromatography. The removal of excess protein may be more complicated. It can be dealt with before the assay by further purification of the sample. Alternatively, the excess can remain during the incubation and be removed after the assay by introducing a termination step to precipitate all proteins, which are then removed by filtration. And finally, proteolytic activities can be eliminated by the addition of the inhibitory cocktail mentioned below. 

F%. 10. Schematic diagram of a radioisoto c method for assaying proteolytic activity... 

Serum albumin labeled with an iodine radionuclide was firstly used as a substrate for determining protease activity by Absolon This method was later on modified several times and applied for assaying various proteolytic activities in different materials. Mego et al. injected denaturated I-human %rum albumin into the tail vein of rats and measured the rate of intralysosomal proteolysis on isolated lysosomes containing endocytosed substrate. This method was also used for the determining the intralysosomal pH on the basis of differences found in the rate of I-albumin breakdown in intact and lysed lysosomes C-bovine serum albumin, I-casein or I-albumin have been alternatively used as substrate for measuring the activity of trypsin, chymotrypsin and papain - ). 

The dyed proteins, though also not very cheap, are much more suitable from an economical point of view. The use of insoluble dyed proteins (e.g. hide powder azure) is very simple and handy. After the chosen reaction time the insoluble protein is removed by filtration or centrifugation and the absorbance of the filtrate is immediately measured. For instance, Rinderknecht proved that it was possible to determine trypsin activity with HPA (hide powder azure) in ng quantities per ml and proteolytic activities in biological materials, tissue extracts, serum, urine, faeces, etc. Other authors applied this substrate for assaying proteolytic activity in beer stabilized with chillproofing preparations containing proteases (mainly papain)... 

The properties of protein fragments can Ik also used for the fluorometric assay of proteolytic activity. Curoffproposed that the increase in trichloroacetic acid-soluble tyrosine (both free and bound in small peptides) can be fluorometrically assayed after the condensation with nitrosonaphthol. 

Apart from assays of proteolytic activity and immunochemical methods (7.1 and 7.5), protein labeling is exploited in other areas of analysis. Noteworthy among them are protein determination in complex mixtures or even directly in cells, protein separation (affinity chromatography and electrophoresis) and protein detection following various separation techniques. For this purpose different labels are applied. 

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