Pronase solution preparation

If pronase digestion of the biotinylated protein is to be done, heat 100 pi of the sample at 56°C for 10 minutes, then add 10 pi of the pronase solution. Allow the sample to digest enzymatically at room temperature overnight. If no pronase digestion is desired, simply use the biotinylated protein solution prepared in step 3 without further treatment. 

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). 

A 1 % (w/v) solution of Pronase from Boehringer in Ham s F12 is prepared, filtered under sterile conditions, and supplemented with 10 % FCS. 

The first total synthesis of amiclenomycin, an inhibitor of biotin biosynthesis, was completed by A. Marquet and co-workers. In order to prove its structure unambiguously, both the cis and trans isomers were prepared. The L-amino acid functionality was installed by a Strecker reaction using TMSCN in the presence of catalytic amounts of Znla. The resulting O-TMS protected cyanohydrin was exposed to saturated methanolic ammonia solution, which gave rise to the corresponding a-amino nitrile. Enzymatic hydrolysis with immobilized pronase afforded the desired L-amino acid. 

Everted Sac Experiments. Hydrolysate solutions for use in the everted sac experiments were prepared by mixing 15 ml of the thawed pronase hydrolysates with 5 ml of a solution containing 4.50 g/1 NaCl, 0.74 g/1 KQ and 2.10 g/1 NaH003. The solutions were equilibrated with 95% 5% oxygen carbon dioxide at 37 C (pH 8.0) and osmolality (Wescor, Inc., Logan, UT) was observed to be within 10% of 302 mOsm. 

Intestinal Perfusion Experiments. Hydrolysate solutions for use in the perfused Intestine experiments were prepared by concentrating the pronase hydrolysates approximately two-fold in a rotary evaporator at 60 C (to inactivate pronase, 24) and mixing the equivalent of 157.5 mg (untreated zeln), 289.2 mg (Ca(0H)2-treated zein) or 150.0 mg (NaOH-treated zein) with 0.05M HEPES (Calbio-chem-Behring Corp., LaJolla, CA) buffer (pH 8.0). The final solutions had a pH of 8.10 0.05. 

Pronase Prepare a 10 mg/mL stock solution of Pronase (Roche) in ultrapure water, aliquot, and store at -20 °C. 

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