Digestion with pepsin

Reaction of purified Ca " -ATPase with 0.3 mM NBD-Cl in the presence of 1 mM AMP-PNP and 1 mM CaCl2 caused inhibition of ATPase activity with the incorporation of 2= 15 nmol NBD-Cl per mg protein [335]. The inhibition was attributed to the binding of 7-8 nmol NBD-Cl/mg enzyme protein, corresponding to = 1 mol NBD-Cl per mol ATPase. The NBD-labeled enzyme was digested with pepsin and several NBD-labeled peptides were isolated [335]. All peptides contained the Gly-X (Cys) sequence that occurs only in one place in the Ca -ATPase, i.e., at Gly343-Cys344. Therefore NBD-Cl reacts with the same cysteine 344 residue that is also modified by maleimide derivatives [319]. The NBD modified enzyme had only 5-10% of the ATPase activity of the control ATPase, but the steady state concentration of the phosphoenzyme intermediate was only slightly reduced [335]. The Ca ... 

A key future direction is expanding PLIMSTEX to provide a higher resolved view than the global view of the whole protein that is currently obtained. Digestion with pepsin followed by peptide analysis by MS and MS/MS should allow kinetics and titrations to be measured for portions of a protein, giving PLIMSTEX a view of the protein that currently emerges from NMR and X-ray methods. 

Paraffin sections (6 p,m thick) of formalin-fixed tissues are mounted onto silanized glass slides and air-dried at 60°C (Henke and Ayhan, 1994). They are deparaffinized, rehydrated, and air-dried. The slides are placed in aplastic Coplinjar filled with lOmM sodium citrate buffer (pH 6.0) and microwaved (720 W) for 1 min this time is measured after reaching the boiling point. After treating with 1 M sodium thiocyanate for 10 min at 80°C, the sections are washed with distilled water and then digested with pepsin (3mg/ml in 0.2 N HC1) for 6 min at 37°C. Both the heating and the pepsin digestion durations must be... 

Pancreatic enzymes, preferably trypsin, have been used for the chemical characterisation and identification of many known bioactive peptides. For example, ACE-inhibitory peptides as well as CPPs are most commonly produced by trypsin (Maruyama and Suzuki, 1982 Berrocal et al., 1989). On the other hand, other enzymes and different enzyme combinations of proteinases, including alcalase, chymotrypsin, pancreatin and pepsin, as well as enzymes from bacterial and fungal sources have also been utilised to generate bioactive peptides. Higher yields of CPPs and, particularly, higher amounts of asl-casein f(59-79) in the hydrolysate have been obtained with casein micelles successively digested with pepsin and trypsin... 

Ono, T., Takagi, Y., and Kunishi, I. 1998. Casein phosphopeptides from casein micelles by successive digestion with pepsin and trypsin. Biosci. Biotechnol. Biochem. 62, 16-21. 

Fidder and coworkers (50) developed a versatile procedure that identifies phosphylated butyrylcholi-nesterase. Adducted butyrylcholinesterase is isolated from plasma by affinity chromatography (procainamide column), digested with pepsin, and a nonapep-tide containing the phosphylated active-site serine residue detected using LC/ESI/MS/MS (quadrupole-TOF hybrid instrument). A C18 150 x 0.3-mm LC column was used, eluted with a gradient of water-acetonitrile-0.2 % formic acid. The method was applied successfully to casualties of sarin poisoning from the Tokyo subway attack (see Chapter 17). 

Yeast Acylation Lower digestibility with pepsin and pancreatin, decreased emulsion stability, increased viscosity, altered solubility (61, 88)... 

Figure 9. Nerve agent adducts with BuChE and their digestion with pepsin...

White powder. Freely sol in water. Partly precipitated at the isoelectric point (pH 4.65-4.80). Appreciably soluble in 60 to 70% alcohol or acetone. Almost completely precipitat -ed in 2.5% trichloroacetic acid soln. Also precipitated from dilute soln by 20% sulfosalicylic acid and by 5% lead acetate soln. Solns are stable to heat. An aq soln buffered to pH 7.5 may be put in a boiling water bath for at least 120 minutes. In 0.10 molar HC1 an 0.2% soln retains its biological potency when kept at 100° for 60 min, but in 0.10 molar NaOH the activity is lost within 30 min. At 60° at 0.2% soln at pH 10.8 maintains its potency for 60 min. In general the substance is more stable in acid soln. Pure ACTH has 150 to 200 potency units per mg. The hiological activity is not destroyed by digestion with pepsin, and hydrolysis products have the biological activity of ACTH. One U.S.P. unit or one international unit or one Armour unit or one potency unit denotes the same activity. 

The proteins are digested with pepsin (which is active at pH 2.6 and 0 °C) and then analyzed by LC/MS to determine the extent of deuteration and for the calculation of kinetic data. Back exchange still occurs even at pH 2.6 and 0 °C, so the enzymatic digestion and chromatography have to be carried out rapidly, in 10-20 min. Alternatively, instead of digesting the proteins, it is possible to determine the overall extent of deuterium incorporation by using ESI-MS to measure the molecular mass of the intact protein. 

Digestion with pepsin and chromatography (0 "C and pH 2.6) followed by MS, are used to determine the locations of the deuterons. 

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