Protease activity measurement

A protease-specific model has also been reported in which a replication-defective adenovirus encoding an NS3 protease-SEAP fusion protein is injected into mouse tail veins, resulting in expression of the fusion protein in the liver [82, 83]. Protease activity can be detected both by measuring activity of liberated SEAP or by protease-induced liver damage. Protease activity was found to be reduced by administration of protease inhibitors. This model can be used to show that candidate inhibitors have adequate pharmacokinetic properties in mice to function in the intended target organ, but it is not a true disease model. 

A different strategy for measuring protease activity is based on the property of xanthene dyes to form H-type dimers (see Sect. 6.2.3) when they are in close proximity. These dimers are accompanied with a characteristic quenching of their fluorescence and, particularly for rhodamines, with a blue shift in the absorption spectrum [121, 122]. The probe D-NorFES-D designed to measure activity of elastase in HL-60 cells consists of an undecapeptide derivatized with one tetramethylrhodamine dye on each side. The sequence contains proline residues to create a bent structure and bring the two fluoro-phores in close proximity. Intact D-NorFES-D shows 90% of its fluorescence quenched plus a blue shift of the absorption spectrum. After addition of the serine protease elastase, an increase in the fluorescence and a bathochromic shift of the absorption spectrum is observed, resulting in an increase in the emission ratio [80],... 

Jones LJ, Upson RH, Haugland RP, Panchuk-Voloshina N, Zhou M, Haugland RP. Quenched BODIPY dye-labeled casein substrates for the assay of protease activity by direct fluorescence measurement. Anal Biochem 1997 251(2) 144-152. 

Blocking of amine groups on proteins also has been used to create a sensitive reagent for measuring protease activity (Hatakeyama etal., 1992). With nearly all the primary amines of casein blocked, an amine detection reagent such as trinitrobenzene sulfonic acid (TNBS) will only minimally react with the protein and form its typical orange derivative. As proteases cleave the protein, however, primary a-amines are created from cleavage of the a-chain peptide bonds, and TNBS can react with them. The more protease activity present, the more color is formed. 

Levine, L.M. et al. 1997. Measurement of specific protease activity utilizing fluorescence polarization. Anal. Biochem. 247, 83-88. 

The recent identification of selective protease substrates to simultaneously measure markers of both viable and dead cells led to the development of optional methods for HTS that provide flexibility and added advantages (Niles et al. 2007a). The assay to measure viable cells is based on a cell-permeable protease substrate called glycyl-phenylalanyl-aminofluorocoumarin (GF-AFC). The procedure is a homogeneous add-incubate-measure method that is faster, more sensitive, and less toxic to cells than the tetrazolium and resazurin reduction assays. The substrate can be prepared in an aqueous buffer and is added directly to samples containing cells. The substrate permeates viable cells where constitutive protease activity in the cytoplasm rapidly removes the amino acids, yielding free AFC. The amount of AFC released is directly proportional to viable cell numbers and shows improved sensitivity compared to the resazurin assay (Figure 6.5). The AFC is detected via a microplate fluo-rometer equipped with a (380- to 400-nm excitation/505-nm emission) filter set. 

A major advantage of this method is the ability to multiplex with other assays. The GF-AFC substrate used to detect viable cells was designed for use in combination with another substrate that selectively detects protease activity from dead cells (Niles, Moravec, and Riss 2008). The method used to measure dead cells is based on the /riv-Ala-Ala-Phe-rhodaminc 110 (AAF-R110) protease substrate. This substrate is non-permeable thus viable cells do not substantially contribute to signal. Dead cells with compromised membranes leak protease activity into the surrounding medium... 

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 - ). 

Protease assay CMV protease activity was measured using the surface plasmon resonance technology of the BIAcore instrument (Pharmacia Biosensor). Briefly, this instrument was used to quantitate residual uncleaved biotinylated... 

Because of the heterogeneous nature of methloninase catalysis (Figure 1), measurement of alpha-ketobutyrlc acid (as well as other carbonyls that may be present) may not provide accurate Indications of the activity of the enzyme. Comparison of the data in Figure 2 for cell-free extracts and semi-purified freeze dried methloninase solutions suggests that either protease activity was involved or that complex Interactions of potential substrates and products occurred. Use of alpha-ketobutyrlc acid assays for methloninase activity in samples Incubated for prolonged periods seems potentially vulnerable to such analytical pitfalls. 

Compound A was tested by using two assays (1) direct inhibition of HIV protease in vitro and (2) inhibition of viral RNA production in HIV infected cells, a measure of viral replication. The results of these assays are shown here. The HIV protease activity is measured with a substrate peptide present at a concentration equal to its value. 

The reaction was terminated by adding 2 ml of 5% trichloroacetic acid to each tube. The tubes were centrifuged for 15 minutes at 25,000 rpm and the optical density of the supernatant was measured at 366 nm. It was found that the measured protease activities were linearly proportional to the amount of enzyme. 

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