Trypsin assay

C9. Crossley, J. R., Smith, P. A., Edgar, B. W., Gluckman, P. D., and Elliott, R. B., Neonatal screening for cystic fibrosis, using immunoreactive trypsin assay in dried blood spots. Clin. Chim. Acta 113, 111-121 (1981). 

Based on these findings, two new classes of potent thrombin inhibitors have been discovered by a three-step process [6-8]. First, a collection of small, basic molecules has been tested in thrombin and trypsin assays iV-amidinopiperidine has been selected as being more active than benzamidine and slightly selective for thrombin. Secondly, substituents attached to the 3-position of amidinopiperidine containing two hydrophobic moieties mediate low nano-... 

Treatment of -ABSC-HEMA with glutaraldehyde produced enzyme supports capable of binding up to 55 wt % trypsin. Incorporation of hydrophobic styrene units Into the support reduced the capacity to 2-. 4 wt X but enhanced the specific activity of the trypsin. The esterase activity of bound trypsin, assayed with TAME, was found to range from 11% to 45% of that exhibited by the free trypsin. Active-site titration of a PHEMA-trypsln conjugate with p-nltrophenyl-p -guanadlnobenzoate HCl Indicated the active species to be 31% of the total amount of protein bound. 

Due to their response mechanism the polyion-selective electrodes are not sensitive to the small fragments of polyionic macromolecules. Thus, if an enzyme cleaves the polyionic molecule these sensors can be used for detection of enzyme activity. Polycation protamine is rich in arginine residues that make it a suitable substrate for protease-sensitive electrochemical assays. Real-time detection of trypsine activity was demonstrated with the protamine-selective electrode as a detector [38],... 

The trypsinized cultures are counted and a sample is assessed for survival as for the cytotoxicity assay. In addition, an appropriate number of cells are reseeded for estimation of mutation frequency at the day 8 expression time. The cells are transferred to roller bottles (usually 490 cm2) for this stage. The bottles are gassed with pure CO2, the tops are tightened and the bottles are incubated at 37°C on a roller machine (approximate speed 0.5-1.0 rev min-1). Usually 106 7 8 9 viable cells are reseeded in 50 ml of Eagle s medium containing serum, but more cells are required at the toxic dose levels. 

The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and Gl. The procedure is based on the fact that the fluorescence quantum yield of cis-parinaric acid increases 40-fold when cis-parinaric acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a picryl sulfonic acid (TNBS) assay (28). 

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... 

HCT-116 human colon carcinoma (ATCC, Bethesda MD) cells were grown in McCoy s 5A) and were routinely subcultured twice weekly. Antiproliferative assay was performed by chemoluminescence assay based on quantification of ATP. Cells in their exponential phase of growth were treated at different times (lh or 24h) with different concentrations of edotecarin or SN-38. For post-treatment recovery studies, cells were washed with PBS and left in drug-free culture medium. Then, cell medium was collected to avoid any cell loss. Cells in monolayer were washed, detached with trypsin, and collected in the medium. Cells were counted in a Multisizer 3 Coulter Counter to measure the drug s effects on growth inhibition. Samples were fixed either... 

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]. 

Collagenase assays were carried out in 50 mM Tricine, 0.2 M NaCl, 10 mM CaCl2 (pH 7.5) containing 0.05% Brij-35 and 50 //M ZnS04 at 30 C on p-chloromercuribenzoate-activated HNC and trypsin-activated HFC using rat type I collagen as substrate. a-Ms denote a-... 

Marcotte, P.A., Richardson, P.L., Guo, J., Barrett, L.W., Xu, N., Gunasekera, A. and Glaser, K.B. (2004) Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction. Analytical Biochemistry, 332, 90-99. 

Fig. 7. A representative structure from a series of 72 compounds each of which was assayed against thrombin, trypsin, and factor Xa.

We now consider some applications of the VSA descriptors to receptor affinity modeling. Figure 7 depicts a typical structure of a series of 72 compounds each of which has been assayed against each of thrombin, trypsin, and factor Xa (27). The PEOE VSA, SlogP VSA, and SMR VSA descriptors were calculated... 

Evidence demonstrating that the degradation observed is catalyzed by enzyme(s) was obtained by typical denaturing treatments. Late extracellular protein fractions from both strains present the same characteristics resistance to heat up to 100°C, partial resistance to acidity as low as pH 1.0 (samples being returned to pH 7.8 prior to assaying for activity), but are completely inactivated by proteolysis with a mixture of trypsin and chy-motrypsin (Table II). 

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