Chymotrypsin solution preparation

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 ... 

Preparation of Chemically Modified Chymotrypsin. Chemically modified chymotrypsin (12) was prepared by following methods Z-DSP (2.5-20 equivalent to the amino groups of chymotrypsin) was added to the buffer solution (Na2B407-Ha, pH 8) of chymotrypsin (14mg). The solution was allowed to stand for 16 hours in refrigerator. And the solution was centrifuged (lO.OOOrpm, lOmin). The solution was (tialyzed, and obtained chemically modified chymotrypsin by lyophilization. 

Figure 1.2 Water adsorption isotherms at 25°C for Celite and preparations obtained by mixing Celite with different solutions (l.OmLg 1 Celite) and drying. Pure Celite ( ) Celite and a-chymotrypsin in water (4.0mgmD )(D) Celite and a-chymotrysin (4.0mgmL l) in 50mM sodium phosphate buffer, pH 7.8 (O). Reprinted from Ref. [18].

Catalytic activities of a-chymotrypsin and Subtilisin Carlsberg in various hydrous organic solvents were measured as a function of how the enzyme suspension had been prepared (Ke, 1998). Direct suspension of the lyophilized enzyme in the solvent containing 1% water was compared with precipitation of the same enzyme from its aqueous solution by a 100-fold dilution with anhydrous solvent. The reaction rate in a given non-aqueous enzymatic system was found to depend on the nature of both enzyme and solvent, but to depend strongly on the mode of enzyme preparation. 

For many years a solution of crude acetone powder of bovine or porcine pancreas has been used to disaggregate tissues and to release cultured cells from their substratum. These preparations — referred to as trypsin 1 250 (based on an international standard) — contain not only trypsin but a range of enzymes including chymotrypsin and elastase which are equally important. Purified trypsin seldom is as efficient as the cruder preparations, especially on tissue disaggregation, when mucinous clumps result (Ronaldoni, 1959). 

Another immobilization method was described by Maeda and coworkers [344], They developed a facile and inexpensive preparation method for the formation of an enzyme-polymeric membrane on the inner wall of the microchannel (PTFE) through cross-linking polymerization in a laminar flow. With this approach, a-chymotrypsin was immobilized successfully. The activity of the immobilized enzyme was tested using N-glutaryl-L-phenylalanine p-nitroanilide as substrate, and the reaction products were analyzed offline by HPLC. There was no significant difference in the hydrolysis efficiency compared to solution-phase batchwise reactions using the same enzyme/substrate molar ratio (Scheme 4.87). 

Sample Preparation Dissolve a sufficient amount of sample, accurately weighed, in 0.001 N hydrochloric acid to produce a solution containing between 12 and 16 USP Chymotrypsin Units per milliliter. This solution should cause a change in absorbance between 0.008 and 0.012 in a 30-s interval. 

Chymotrypsin from bovine pancreas is dissolved (31.8 mg/mL) in 50 mM sodium phosphate buffer pH 7.8, and immobilized by drying onto the support. Fifty microliters of enzyme solution is mixed with 50 mg of celite. The preparation is dried under vacuum overnight. The immobilized enzyme preparadon is added to an organic solvent (like hexane, chloroform, or toluene). Substrates are dissolved in the organic phase. Water must be added to obtain enzyme acdvity, but the amount of water present must be carefully controlled. 

Trypsin preparations are often contaminated by chymotrypsin, which can be inactivated by treatment with L-(l-tosylamino-2-phenyl)ethylchloromethyl ketone (TPCK). Trypsin in solution should be stored in 10 mM HC1, otherwise it will undergo autoproteolysis. For proteolytic cleavage, trypsin is dissolved (freshly) in 100 mM Na bicarbonate buffer and added in a proportion of 2-10% (w/w) of the protein substrate. Reaction can be stopped by addition of phenylmethylsulphonyl-fluoride (PMSF) or soybean trypsin inhibitor (4 mg inhibitor per mg trypsin). 

Both peptides have been prepared in solution for the last 30 years (136, 137), and many solid-phase syntheses also have been reported. In addition, syntheses of these peptides have employed enzymatic methods, which could be done in low-water content systems on a preparative scale (138). A"-Cbz and A -Boc protected Met-enkephalin and Leu-enkephalin were prepared by means of a-chymotrypsin, papain, thermolysin, and bromelain adsorbed on Celite. a-Chymotrypsin has a primary specificity for bulky and hydrophobic amino acids (Phe, Tyr,... 

Stock solutions of DFP can be conveniently prepared in isopropanol in concentrations from 0.1 M to 0.001 M. These solutions are stable for a month in the refrigerator (Jansen et al. 1949). Moon et al. (1965), in their studies of the reaction of chymotrypsin with DFP, have determined the normality of stock solution of DFP in the following manner. Roughly 0.03 moles of DFP were added to an aqueous solution of 0.17 moles of KOH in a volumetric flask. The solution was allowed to stand for 12 hr at 25°C to permit complete hydrolysis, and was titrated to pH 7.0 with 0.1 N HCl. Identical titrimetric results were obtained after 43 hr standing in alkali indicating that complete hydrolysis had taken place after 12 hr. To determine the amount of free acid, if any, in the DFP, the same amount of DFP that was used... 

N-(m/is-Cinnamoyllmidazole (1). Mol. wt. 198.22, m.p. 133-133.5°. Prepared in high yield by reaction of cinnamoyl chloride with imidazole in benzene at 10-25°. The reagent reacts rapidly and quantitatively with the active site of a-chymotrypsin and hence can be used for the spectrophotometric determination of the normality of an enzyme solution by titration. 

Few medicines based on boron are known, in general boric acid or a boronic acid serve to esterify an a-diol or an o-diphenol. This is the case for the emetic antimony borotartrates of the ancient pharmacopoeias, for the injectable catecholamine solutions, for tolboxane, which is close to meprobamate and which was commercially available as a tranquillizer some decades ago, and also for the phenylboronic esters of chloramphenicol. Boro-mycine was the first natural product containing boron. It is a complex between boric acid and a polyhydroxylated tetradentate macrocycle. Some boronic analogues of amino acids were prepared as chymotrypsine and elastase inhibitors. The most important medical use of derivatives of boron derivatives is the treatment of some tumours by neutron capture therapy, " the problem here being to ensure a sufficient concentration of the product in the tumour being treated. 

Other protein-rhodium conjugates containing cationic rhodium catalysts have also been prepared using bis(diphenylphosphino-ethyl)amino derivatives 5-7 and solutions of these bis(phosphine) ligands in the presence of carbonic anhydrase, a-chymotrypsin and bovine serum albumin (47). However, the exact nature of the complexes formed has not been discerned in any of these cases, and these latter enzyme-transition metal complexes evidently do not exhibit enantioselectivity in hydrogenation of a-acetamidoacrylic acid. 

Enzyme stock preparations To make 1 ml trypsin, chymotrypsin, or endoproteinase Glu C stock solution, dissolve 1 mg enzyme in 1 ml UHQ water. Distribute the solution in 50-//1 portions in 0.5-ml Eppendorf tubes, lyophilize in a vacuum centrifuge, and store at —20°C. To make 0.2 ml endoproteinase Asp-N stock solution, dissolve 10 //g enzyme in 200 //I UHQ water. Distribute in 10-/xl portions, lyophilize, and store as above. To make 100 //I carboxypeptidase Y, A, or M stock solution, dissolve 100 //g enzyme in 100 //I UHQ water. Distribute in 10-(A portions, lyophilize, and store as above. 

Self-test 8.1 j The enzyme a-chymotrypsin (Atlas P3) is secreted in the pancreas of mammals and cleaves peptide bonds made between certain amino acids. Several solutions containing the small peptide N-glutaryl-L-phenylalanine-p-nitroanihde at different concentrations were prepared, and the same small amount of a-chymotrypsin was added to each one. The following data were obtained on the initial rates of the formation of product ... 

Quantitative modification of tryptophan in proteins can be achieved readily by using a low molar ratio of reagent to protein. The tryptophan residues of lysozyme, trypsin, and chymotrypsin were fully modified by reacting the protein with 20 equivalents of a freshly prepared solution of 2-nitrophenylsulfenyl chloride in 50% acetic acid (354). Acetate buffer pH 4-5 has been used in the modification of human growth hormone (60), lysozyme (341) and a-lactalabumin (357). 

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