Proteases solution preparation

The higher molecular mass contaminants in conventional insulin preparations include various proteases. Such preparations are generally maintained in solution at acidic pH values (often as low as pH 2.5-3.5). This minimizes the risk of proteolytic degradation of the insulin molecules, as contaminant proteases are inactive at such pH values. 

Protease Solution Use a freshly prepared solution containing 50 mg of protease (Sigma Chemical Co. catalog number P 3910, or equivalent) per milliliter of Mixed 8.2 Buffer Solution. 

Complete protease inhibitor tablets (Roche, Mannheim Germany) solubilized in water to IX immediately before use. Individual protease inhibitors prepared as stock solutions benzamidine (Sigma) 0.1 M in water stored at -20°C and used in buffers 1,2, and 3 at 1 mM final concentration TLCK (Roche) 1 mg/mL in 0.05 M sodium acetate pH 5.0 (Sigma) stored at -20 C and used in buffers 1,2, and 3 at 50 pg/mL final concentration TPCK (Roche) 3 mg/mL in ethanol stored at-20°C and used in buffers 1,2, and 3 at 50 Xg/mL final concentration pepstatin (Roche) 1 mg/ mL in ethanol stored at -20°C and used in buffers 1,2, and 3 at 1 (ig/mL. 

Make protease dilutions in lx TNG buffer see Note 16 for protease choice and solution preparation). Be sure to use a fresh aliquot of protease in every experiment. 

Add 2 pL of the range of protease solutions see Note 16 for protease choice) prepared in Subheading 3.1, step 13, to achieve the appropriate final ratio of total enzyme to total substrate in each sample. Add the protease solutions at specific intervals (e.g., every 30 s) to ensure that each sample is digested for the same amount of time. Be sure to include a sample that is not digested. For the non-digested sample, add 2 pL of lx TNG buffer instead of protease. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

Because PMSE fails to inactivate acetylcholinesterase, this reagent is much less toxic than diisopropylfluoro-phosphate, and is also recommended as an alternative to the neurotoxic fluorophosphates and fluorophospho-nates. PMSE is freshly prepared as a 1-3 mM solution in water (higher concentrations will precipitate spontaneously). A better procedure is to first prepare a 20 mM PMSE solution in 2-propanol or dioxane this solution can then be added to the biological fluid with vortex mixing to achieve a 1-3 mM final concentration as a homogeneous solution. One should confirm that the alcohol or dioxane has little or no undesirable effect on enzymes or proteins of interest. See Chymotrypsin Protease Inhibitor Cocktails ... 

Of course, the goal of every synthetic organic chemist is to obtain crystalline products, and a few cases of crystalline synthetic proteins have been reported. These include the ribonuclease A synthesized in solution/35 which crystallized after chemical workup and affinity chromatography (see Section 5.1.6.2.2). Further examples include an HIV protease analogue/701 a ubiquitin analogue/59 and monellin/89 which were each prepared by solid-phase methods and purified by HPLC. 

An alternative to the synthesis of proteins by classical fragment synthesis in solution or by solid-phase synthesis on a support is the use of enzyme-catalyzed condensation of amino acids or peptides. This possibility was first demonstrated in 1938 91 with the synthesis of poorly soluble benzoyl-leucyl-leucine anilide by papain catalysis. After many years, this approach was extended to the preparation of peptide hormones such as Leu-enkephalin 92 and dynorphin(l -8).[93 This was made possible by the use of highly purified enzymes and by careful control of reaction conditions. The basic principles of protease-catalyzed peptide bond formation have been discussed.194 ... 

Reduction of the carbonyl in the r >[CO-CH2-NH] link 7 (R1 = H) results in the (hy-droxy)ethyleneamino or r >[CH(OH)-CH2-NH] link 8 (R1 = H), which has proved to be a very potent analogue of the tetrahedral hydrated intermediate of the scissile amide bond. It has been widely used for the design of various inhibitors of HIV protease 141,142 14 154 and ACE, 155-157 and to synthesize angiotensin II, III, and IV analogues. 158,159 Indeed, the chirality of the hydroxylated carbon is critical for HIV protease inhibition, but separation of the epimers may be difficult. Therefore, the stereoselective synthesis from epoxides has been actively investigated. An example of a C-methylated tp[CMe(OH)-CH2-NH] link, obtained from an epoxide with chromatographic separation of the epimers, has also been described. 157 Most of the [(hydroxy)ethyleneamino] peptides have been prepared by solution procedures, but two examples of solid-phase synthesis have been reported. A theoretical study of the (hydroxy)ethyleneamino replacement for the amide bond has been carried out on a HIV protease inhibitor. 160 ... 

A similar strategy was used to examine the potential role of a reverse turn as a recognition element adjacent to the cleavage site of substrates of HIV protease.197 A series of inhibitors was prepared, the synthesis of which involved the solution coupling of the statine-like transition state mimic to the (3-turn mimetic to provide 46 (Scheme 22). Subsequent sodium in ammonia reduction provided analogue 47. One of the compounds was a reasonably potent inhibitor of protease activity (IC50=2.6 x 10-8 M) (Table 1). 

Peptide a-oxo acids, a-oxo esters, and a-oxoamides are also potent inhibitors of cysteine and serine proteases. Oxidation of peptide a-substituted carboxylic acid derivatives provides a general route to these compounds (Section 15.1.5). Peptide hydroxamic acids have been shown to be inhibitors of metalloproteinase and some have been reported to have antibiotic, anticarcinogenic, and antiviral activities. Peptide hydroxamic adds may be prepared by solution and solid-phase methods using a variety of resins (Section 15.1.6). a-Aminoboronic acids may be prepared by several routes and are reported to be inhibitors of aminopepti-dases. Procedures have been developed for their incorporation into peptides (Section 15.1.7). 

Degradation could be due to the presence of proteases or to inherent low enzyme stability. For prevention, a cocktail of protease inhibitors can be added (e. g., Complete tabs, Roche, Penzberg, Germany). Always use freshly prepared phage solutions when performing selection from libraries. 

Proteomics in parasitic flatworms can be completed on intracellular fractions (e.g. microsomal or cytosol) or at the host-interface on excretory-secretory (ES) products. ES analysis can be completed during in vitro culture or in vivo by, for example, bile or gut content analysis. In all cases, a rapid and careful preparation is vital to prevent altered pro-teomic profiles due to stress responses (upreg-ulation of heat shock proteins) and action of proteases. Parasitic flatworms are best extracted from fresh host material, washed with a buffered saline solution at approximately the host s body temperature. In F. hepatica, for example, this will allow regurgitation of gut contents to remove digested material from, and removal of host material adherent to the outer surfaces of the parasite (Jefferies et al., 2001), both of which can subsequently complicate separation and identification. 

Once bearing some substituents, the decrease of polarity of the sucrose derivatives makes them soluble in less-polar solvents, such as acetone or tert-butanol, in which some lipases are able to catalyze esterifications. Unlike proteases, which necessitate most often the use of an activated acyl donor (such as vinyl or trifluoroethyl esters), lipases are active with simple esters and even the parent carboxylic acids in the presence of a water scavenger. The selectivity of the lipase-catalyzed second esterification is specific for OH-6 allowing the synthesis of mixed T,6 -diesters.123,124 For some lipases, a chain-length dependence on the regiochemistry was observed.125 Selectively substituted monoesters were thus prepared and studied for their solution and thermotropic behavior.126,127 Combinations of enzyme-mediated and purely chemical esterifications led to a series of specifically substituted sucrose fatty acid diesters with variations in the chain length, the level of saturation, and the position on the sugar backbone. This allowed the impact of structural variations on thermotropic properties to be demonstrated (compare Section III.l).128... 

The iotai proteolytic activity of pancreas powder is determined by comparing the quantity of peptides nonprecipitabie by a 556 m/V solution of trichloroacetic acid R released per minute from a substrate of casein solution with the quantity of such peptides released by pancreas powder (protease) UK from the same substrate in the same conditions. For the test suspension and the reference suspen-sion, prepare the suspension and carry out tiie dilution at (W-°C. 

Enzymatic gelation of partially heat-denatured whey proteins by trypsin, papain, pronase, pepsin, and a preparation of Streptomyces griseus has been studied (Sato et al., 1995). Only peptic hydrolysate did not form a gel. The strength of the gel depended on the enzyme used and increased with increasing DH. Hydrolysis of whey protein concentrate with a glutamic acid specific protease from Bacillus licheniformis at pH 8 and 8% protein concentration has been shown to produce plastein aggregates (Budtz and Nielsen, 1992). The viscosity of the solution increased dramatically during hydrolysis and reached a maximum at 6% DH. Incubation of sodium caseinate with pepsin or papain resulted in a 55-77% reduction in the apparent viscosity (Hooker et al., 1982). 

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