Trypsin films

A model that is consistent with these observations of the action of trypsin and phospholipase A and with the discontinuities in the All-composition curves (Figures 2 and 3) is one in which the lipid monolayer is not a continuous palisade of uniformly oriented lipid molecules but rather an assembly of surface micelles. In this model, proposed by Colacicco (4, 5), the protein first comes into contact with the lipid molecules at the periphery of the surface micelles and then inserts itself as a unit between them. This is the basis for the generalized nonspecific interaction between lipids and proteins which results in increase of surface pressure. One may thus explain the identical All values obtained with films of lecithin and 80 mole % lactoside by picturing the lecithin molecules outside and the lactoside molecules inside the surface micelles. In this model lecithin prevents the bound lactoside from interacting nonspecifically with globulin and produces the same increase in pressure as with a film of pure lecithin. In the mixed micelle the lactose moiety of the lactoside protrudes into the aqueous subphase. Contact of the protein with these or other nonperipheral regions of the surface micelle would not increase the surface pressure. 

Examples of studies of local conformational dynamics include the films made by Richard Feldmann, in collaboration with M. Levitt and with M. Karplus, which show the dynamics of pancreatic trypsin inhibitor and its interaction with solvent, and the study by Case and Karplus of the pathway by which an oxygen molecule can enter and leave the binding pocket of myoglobin (31). (In the static structure, there is no stereochemically feasible path for binding oxygen — the process requires a distortion of the protein structure.)... 

Upon the compression of films formed by globular proteins (such as albumine, globuline, hemoglobin, trypsine, and others) up to a pressure of 20 mN m"1 the two-dimensional pressure isotherms are quite reversible. At somewhat higher compression, such that the area per amino-group reaches... 

Figure 33. Detailed biomesogen dynamics treatment rotational isomerizations of Tyr-35 of bovine pancreatic trypsin inhibitor mediated by structural changes in the surrounding biomesogenic protein matrix -overall view (top) and film in skeleton presentation (bottom) [77b-d],...

Samples of Mater Bi ZlOlU/C film, in a size of 15x2cm, and Mater Bi YIOU plate, were placed in a test tube containing trypsin (from bovine pancreas, Wytwomia Surowic i Szczepionek, Warsaw) solution and incubated in the dark in incubator at 37 °C for 3 months. 

The cytotoxicity assay is usually performed by determining the viability of suitable cell lines in the presence of polymers. For this test, 3-5 mm discs of polymer film are cut and sterilised under standard conditions (at 121 C and 6.8 kg (15 lb) pressure for 15 min). The cell growth in the presence of the polymer films is measured under a controlled atmosphere (CO2 incubator, 37"C) using an appropriate culture medium, supplemented by 10% fetal bovine serum and penicillin-streptomycin antibiotic solution. Confluent monolayers are propagated by trypsinisation (0.25% trypsin and 0.02% EDTA, ethylene diamine tetraacetic acid) and re-plated at 2 x 10 cells/mL in a sterile polystyrene cell culture plate, then incubated for 24,48 and 72 h. The morphology of the cells is analysed by light naicroscopy (Leica) after... 

Wait until clearing zones become visible, then stop the reaction by slowly filling the petri dish with cold water. Remove the film from the petri dish, rinse several times with running water, and air-dry. Examples with subtilisin BPN and trypsin are shown in Figs. 1 and 2. 

The substrate was composed of a dried film of bovine serum albumin (BSA) deposited on glass. The etching enzyme was trypsin, a proteolytic enzyme that cleaves on the carboxyl side of lysine and arginine residues. The enzyme was loaded in solution into a nano-fountain pen (NFP) probe (a capillary, heat-drawn into a sharp tip with an aperture of 100 nm) [2] mounted as the probe of an AFM, and delivered in... 

The following experiments made with films of trypsin showed that inactivation had occurred on spreading (14). As will be seen later, spread films of antigens deposited on slides retain their ability to react specifically with homologous immune sera. However, a drop of trypsin solution at a pH above 7 destroys this specific property of the film. If a film of trypsin is first deposited on the slide and then the film of antigen, no inactivation of the antigen film occurs even when a drop of... 

Trypsin can be measured semiquantitatively by its ability to hydrolyse the gelatin on the surface of X-ray film. A number of serial dilutions of the specimen are made and these are spotted onto unexposed X-ray film. Gelatin hydrolysis results in the formation of clear areas on the film, the greatest dilution of the film giving a clear area being an indication of the amount of trypsin present. 

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