Papain assay

Specimens for assay were prepared by suspending 500 mg of lyophilized brain in 50 ml of the buffer-papain mixture and hydrolyzing overnight at 37° C. The solution was then autoclaved for 10 minutes. This procedure yielded higher results than autolysis or prolonged enzymatic hydrolysis. After centrifuging off the debris, 0.5, 1.5, and 2.5 ml of the supernatant were used for assay. This represents 1, 3, and 5 mg of the original brain tissue per milliliter of final medium. 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

In a subsequent report, however, the thiazolopapains were shown to be competent in catalyzing a carbon-carbon bond-forming reaction of the acetoin condensation type (Fig. 14) [50]. The reaction of the papain derivatives with 6-oxo-heptanal was assayed at neutral pH (Fig. 14). The course of the reaction was monitored by HPLC and the products analyzed by H NMR. In the case of the... 

The hbraries of enzyme substrates were obtained by spht-pool synthesis to yield one-bead-one-compound hbraries. The substrate assay was performed with a range of proteolytic enzymes such as subtilisin Carlsberg [26], cruzipain [27], protein disulfide isomerase [28-29], matrix metalloprotease MM P-9 [30], papain [31],... 

Figure 1. Elution profile of CBH I-papain reaction products on two Superose HPSEC columns (HR 6 12) connected in series. Ilte elution of the tail fragment was confirmed using the phenol-sulfiiric acid assay for carbohydrate.

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. 

Calculation One papain unit (PU) is defined in this assay as that quantity of enzyme that liberates the equivalent of 1 p,g of tyrosine per h under the conditions of the assay. 

Papain is a proteolytic enzyme from plants. In the HPLC assay developed to measure its activity, the water-soluble N-benzyl-L-arginine ethyl ester (B AEE) is used as the substrate. 

The reaction mixture contained the substrate BAEE, mercaptoethanol, EDTA, and sodium chloride. The reaction was started by the addition of papain, and the immediate adjustment of the incubation mixture to pH 6.2. After 5 minutes, the reaction was terminated by the addition of 30% acetic acid. Precipitated material was removed by filtration, using a 0.45 /urn Millipore filter, and the filtrate was analyzed by HPLC. The results of an assay are... 

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)... 

Dissolve a suitable amount of papain F.I.R standard in bidistilled water and adjust to pH 7.0 to obtain a solution containing approximately 3 F.I.R units/mL. This solution is kept in ice water during the assay. 

CPTI activity appeared to be reciprocally lowered when glycerol-S hosphate was added to the medium over the range of palmitoyl-CoA / albumin ratios assayed (Fig. 6). This addition should initiate papain-sensitive and insensitive GPATs as the ratios applied, but the mode of inhibition of CPT I seemed to be independent of GPAT activity, since this inhibition still occured for very low GPAT activities (highest ratios, around 4.5 see Fig. 2). 

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