Pepsin titration

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. 

Iodination of PIR (147) showed 1 residue buried, Tyr 25, and all others iodinated at least to the monoiodotyrosyl form. Pepsin-inactivated RNase also has only one abnormal tyrosyl by titration which is thus assumed to be 25. Iodination of RNase-S is very similar to RNase-A in the early stages (lift). Extensive iodination leads to dissociation of the protein and peptide components. Direct iodination of S-protein indicated that all 6 tyrosyl residues were accessible, in this sense comparable to urea-denatured RNase-A. Substantial structural changes must be involved for both S-protein and PIR if Tyr 97, in particular, is to become susceptible to attack (see Section IV,B,3). 

Figure 11. Terbium fluorometric titration of azoalbumin containing an average of 1.6 EDTA groups per albumin molecule (O) azoalbumin, 0.14mM in 0.1 M sodium citrate, pH 6.5 (X) pepsin-digested azoalbumin, 0.17mM sodium citrate, pH 6.5. Adding terbium(III) to solutions having equal concentrations of native albumin or of pepsin-treated native albumin did not lead to detectable fluorescence under...

The simplest explanation of the difference in group count would be that native pepsin has six uncharged carboxyl groups inaccessible to titration, and that these are exposed during denaturation. 

The first experimental criteria to be met in a spectrophotometric titration are that the absorptivities be time-independent, and reversible. This is so fundamental that it should be obvious, but it still must be stressed. Proteins not infrequently possess marked alkali-sensitivity, as shown by the inactivation of pepsin at pH 7, the hydrolysis of one particular amide linkage in a-corticotropin at pH 9 (Shepherd et al, 1956), and the irreversible inactivation of bacitracin at pH > 7 (Weisiger et al, 1955). 

Aliquots of the pepsin digest are analyzed for a) titrat-able acidity (the number of equivalents of KOH required to titrate a 20 g aliquot containing 5 ml of the pancreatin-bile mixture to pH 7.5), b) nonheme iron concentration, and c) 59ye activity. 

A dialysis bag containing an amount of NaHC03 in 25 ml of water equivalent to the previously determined titratable acidity is added to a 20 g aliquot of the pepsin digest. 

Similar possibilities might appear to exist for zein and pepsin, which also contain relatively large numbers of tyrosine residues. Because zein is insoluble in water, however, it has been titrated only in alcohol-water mixtures (Neuberger, 1934 Cohn, Edsall, and Blanchard, 1934), and pepsin is so unstable in neutral and alkaline solution that its dissociation curve above pH 6 (which must refer to the denatured protein) is known only approximately (Herriott and Northrop, 1934). 

Philpot and Small (1938) present evidence, though not from titration data, that nitrous acid attacks the tyrosine residues of pepsin to yield a diazo derivative of the protein. 

Method of GrUtzner. — Fibrin, reduced to threads as fine as posable, is dipped for several seconds in an ammonlacal solution of carmine, is then dried and is rinsed in pure water. The product, colored red, is dissolved in a solution of pepsin hydrochloride, the more rapidly the richer the liqmd is in enzyme. Therefore, a comparative colorimetric determination is made of the quantity of coloring matter dissolved in a test liquid and in a titrated solution of pepsin, at the end of a given time. The ratio of the shades gives the ratio of the enzymic activities. 

The esophagostomy ensured that no juice was contaminated with saliva. Babkin and Komarov measured the free and total acidity of a sample of gastric juice by titration and then added one or another preparation of alcohol-precipitated, dried hog gastric mucus. They placed Mett tubes in the mixture. After they had removed the tubes, Babkin and Komarov again determined the free and total acidity of the samples, but they did not measure the pH. According to their calculations, 330 mg of dried mucin did reduce the peptic power of the juice, and they concluded that mucus does indeed inhibit pepsin. ... 

Iodination of proteins has produced measurable changes in the titration curves. In zein (280), insulin (125), and pepsin (6) the region of the titration curves usually assigned to the phenolic group of tyrosine (pK=10) is displaced in iodinated proteins in the direction of increased acidity by nearly 2 pH units. This is apparently not true for iodinated globin (299). 

Native electrophoresis of pepsin and hemoglobin on 10% polyacrylamide gel carried out at 48 °C during 90 min, according to the Laemmli procedure, at pH 8.3 (Laemly 1970). Water solutions of all samples of enzyme (pepsin dissolved in water to final concentration of 2 mg/mL) were titrated with HCl to pH 2 and incubated at 37 °C, with addition of different concentrations of Al + ion (1, 5 and 10 mM). The samples were diluted with sample buffer in ratio 1 1 (v/ v) and applied on gel in volume of 20 pL. Visualization was performed with Commassie Brilliant Blue G-250 dye. The gels scanned and processed using Corel Draw 11.0 software package. Quantification of electrophoretic mobility of the molecule is carried out via Rs value, where it is defined by ... 

The synthesis of peptides by enzymatic hydrolysis of proteins was carried out with gas-vortex gradientless bioreactor of type BIOK-022. As enzymes were used pepsin and trypsin. Initial concentration of the protein substrates was 20% on weight. Temperature of the synthesis was 36-40 C. Synthesis was carried out during 1 h. Received peptides molecular weight was 800-1100 g/mol of titration method with formalin [2],... 

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