Pepsin, solution preparation

When it attacks proteins, pepsin liberates tyrosine bound by central bonds at the carboxyl end of the peptide chain, as well as tyrosine at the amino terminal end. At the Rockefeller Institute, M. L. Anson and A. E. Mirsky made liberation of tyrosine the basis of their method for estimating pepsin. They added a pepsin solution to a standard solution of hemoglobin in 0.6 N HCl. Hemoglobin is a substrate easily prepared in large quantities it can be stored without deterioration and it is uniform from one batch to another. The acidity chosen by Anson and Mirsky is well on the acid side of the pH where small variations in pH cause large changes in peptic activity. Anson and Mirsky stopped the reaction with trichloracetic acid and measured the tyrosine in the filtrate by means of the Ciocalteu-Folin phenol reagent. The method was universally adopted. ... 

We confirmed the observation of Dunn a]. (17) that peptide 1-16 reacts with pepsin much more rapidly and completely at 37 than at 25. The turbidimetric assay for pepsin, however, was less consistent at 37 than at the 25 temperature recommended by McPhie. We therefore first incubated the peptide with about 300 ng of pepsin in 1 ml of the pH 5.3 buffer for 15 min at 37, then for 5 min at 25 before adding 2 ml of the diluted milk solution at 25 to start the assay. The standard curve for 50-300 ng pepsin was prepared in exactly the same way and rechecked with each new carton of milk. 

Less than 15 mui before use, prepare a solution of pepsin powder BRP containing 0.5 Ph. Bur. U. per milliliter in dilute hydrochloric add R2 before dilution to volume, adjust to pH 1.6 0.1, if necessary, using 1N hydrochloric add. 

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). 

Crews et al. [81] studied cooked cod by means of a two-step in vitro gastrointestinal enzymolysis. For the first step of sample preparation they employed gastric juice (1 percent m/v pepsin, pH = 2.0, in 0.15 mol l-1 NaCl) at 37°C for 4 h. Afterwards a pancreatin-based mixture was added to the sample solution containing 1.5 percent m/v pancreatin, 0.5 percent m/v amylase, and 0.15 percent m/v bile salts in 0.15 mol l-1 NaCl at pH = 6.9 for a further 4 h at 37°C. The relatively short (8 h) enzymatic activity and the lack of enzymes capable of hydrolyzing proteins directly into amino acids resulted in the identification of inorganic Se (IV) only, as no selenoamino acids could be detected. 

Standard Preparation Dissolve 100 mg of USP Pepsin Reference Standard in 150 mL of Hydrochloric Acid Solution. Use this solution within 1 h. 

Sample Preparation Dissolve 100 mg of the pepsin sample, or an amount of the enzyme preparation that will provide a solution similar to or slightly stronger than the Standard Preparation, in 150 mL of Hydrochloric Acid Solution. Use this solution within 1 h. 

Procedure Pipet 5.0 mL of the Standard Preparation into each of two bottles containing the Substrate Preparation. To two or more additional substrate bottles add graduated aliquots of the Sample Preparation so that one bottle will contain approximately the same amount, and the others will contain successively lesser amounts, of pepsin as is contained in the 5.0 mL of the Standard Preparation, using, for example, 5.0, 4.9, and 4.8 mL. When less than 5.0 mL of the Sample Preparation is used, add sufficient Hydrochloric Acid Solution to make 5.0 mL of combined Sample Preparation plus acid added. At once stopper the bottles securely, invert them three times, and heat in a water bath, maintained at 52° 0.5°, for 2.5 h, agitating the contents equally every 10 min by... 

Fig. 9. Treatment of gastric and colonic mucus with lysozyme. Effect of lysozyme in human tears on viscosity of gastric mucus. To three samples of this preparation, 2 ml each, were added, respectively 0.5 ml distilled water, 0.5 ml solution containing 5 mg crystallized pepsin, and 0.5 ml 50% solution of human tears in water. Incubation at pH 6.5 and 37° C. The mucoproteose content was determined after 24 hours of incubation. From Glass et al. (G50).

Sephadex G-15 was purchased from Pharmacia Biotech urea was purchased from Fisher Scientific D2O and pepsin were purchased from Sigma Chemical Co. All chemicals were used without further purification. IDeuterated urea/D20 solution was prepared by repeated lyophilization. 

Preparation. Method of Van Hasselt. — To obtain a solution of artificial pepsin free from other enzymes, the procedure is as follows The stomachal mucous membraxxes of a hog, well minced, are macerated at ordinary temperature for 10 to 15 days in a 5 per cent solution of NaCl. The filtrate is saturated wi NaCl, and the precipitate, rich in coagulating enzyme, is removed. The filtrate, dialyzed to free it of the greater part of the NaCl which it contains, is then saturated with ammonium sulphate. The precipitate obtained, redissolved... 

The experiments of Zunz relate to the products of transformation of egg-albumin, serum-albumin, serum-globulin, as well as casein the first three of these products were in a state of almost absolute purity. The following is the procedure dissolve 2 g. of albuminoid substances in 100 c.c of liquid containing 30 eg. of hydrochloric add and 4 eg. of pepsin. Let it digest at 50°, until a peptone reaction is found. At this point, the hquid is filtered, exactly neutralized, again filtered, and is then acidified with sulphuric add to a dilution of o 75 per cent of the liquid. The salt solution is prepared by saturating in the cold, it has a density of i 450. 

Method of Efihont. — This method permits the estimation in a qui(h and accurate manner of the dissolving power of a pepsin. First of all, an albmnin emulsion is prepared by dissolving 8o g. of dried commercial white of egg in two liters of water. This solution is exactly neutralized, 20 c.c. of HCl (iV)... 

Mefliod of Fuld with Edestin. — Edestin, taken from hemp, can serve as a substrate for the rapid determination of the peptonizing power of pepsin. The following procedure is recommended. Preparation of solutions ... 

Method of Jacoby. — This method is based on the same principle as that already described with regard to the analyas of pepsin. First of all an emulsin of ricin is prepared containing I g. of this substance and 1.5 g. NaQ in 100 c.c. of water. Pour 2 c.c. of this liquid in a series of test tubes, in which is then added o, o.i, 0.2, 0.3, 0.5, 0.7, i.o c.c. of a i per cent solution of trypsin. Then bring all the volumes to 3 c.c., and add in each tube 0.5 c.c. of i per cent NaOH. Allow to stand at 37 , and note the time required for the liquid to become clear. With o.t c.c. of I per cent trypsin of good quality, a complete clarification is obtained at the end of 6 hours. 

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