Trypsin determination

Figure 39 presents an example of trypsin determination. The first and third curves are controls, the second and fourth are tests of respective activities 10 and 100 ng of crystalline trypsin per 1 ml. 

Figure 39. Example of trypsin determination in duodenal juice.

A.A. Kossiakoff, J. Shpungin and M.D. Sintchak, Hydroxyl hydrogen conformations in trypsin determined by the neutron diffraction solvent difference map method Relative importance of steric and electrostatic factors in defining hydrogen-bonding geometries, Proc. Nat. Acad. Sci., USA, 87 (1990) 4468. 

Fig. 15.23. pH activity curves of native (O) and complexed trypsin ( ) determined at 30 °C by using a 2.66 mM BANA solution. Relative activity was calculated from the initial rate of BANA hydrolysis, and 100% activity refers to the hydrolysis rate (4.61 x 10 mM min" by the native enzyme at pH 8.18. (from Ref. [57])... 

To the remainder of the casein solution add 0 5 to o 8 g. of finely powdered commercial trypsin, shake to dissolve, and place in a thermostat (or in an incubator) at 40 . After 15 minutes, remove 25 ml. and add a few drops of phenolphthalein it will now be found that the solution remains colourless. Run in carefully Mj 10 NaOH solution until the colour of the solution is just pink, add 5 ml, of neutralised formalin and then titrate against Mj 10 NaOH solution until the pink colour is just restored note the amount required. Remove fiirther quantities (rf 25 ml. at intervals which must be determined by the speed of the reaction. The following will probably make a suitable series i, 2, 3,... 

Figure 2.14 shows examples of both cases, an isolated ribbon and a p sheet. The isolated ribbon is illustrated by the structure of bovine trypsin inhibitor (Figure 2.14a), a small, very stable polypeptide of 58 amino acids that inhibits the activity of the digestive protease trypsin. The structure has been determined to 1.0 A resolution in the laboratory of Robert Huber in Munich, Germany, and the folding pathway of this protein is discussed in Chapter 6. Hairpin motifs as parts of a p sheet are exemplified by the structure of a snake venom, erabutoxin (Figure 2.14b), which binds to and inhibits... 

Even though these enzymes have no absolute specificity, many of them show a preference for a particular side chain before the scissile bond as seen from the amino end of the polypeptide chain. The preference of chymotrypsin to cleave after large aromatic side chains and of trypsin to cleave after Lys or Arg side chains is exploited when these enzymes are used to produce peptides suitable for amino acid sequence determination and fingerprinting. In each case, the preferred side chain is oriented so as to fit into a pocket of the enzyme called the specificity pocket. 

The results of experiments in which the mutation was made were, however, a complete surprise. The Asp 189-Lys mutant was totally inactive with both Asp and Glu substrates. It was, as expected, also inactive toward Lys and Arg substrates. The mutant was, however, catalytically active with Phe and Tyr substrates, with the same low turnover number as wild-type trypsin. On the other hand, it showed a more than 5000-fold increase in kcat/f m with Leu substrates over wild type. The three-dimensional structure of this interesting mutant has not yet been determined, but the structure of a related mutant Asp 189-His shows the histidine side chain in an unexpected position, buried inside the protein. 

Definition of Ej and E2 eonformations of the a subunit of Na,K-ATPase involves identification of cleavage points in the protein as well as association of cleavage with different rates of inactivation of Na,K-ATPase and K-phosphatase activities [104,105]. In the Ei form of Na,K-ATPase the cleavage patterns of the two serine proteases are clearly distinct. Chymotrypsin cleaves at Leu (C3), Fig. 3A, and both Na,K-ATPase and K-phosphatase are inactivated in a monoexponential pattern [33,106]. Trypsin cleaves the E form rapidly at Lys ° (T2) and more slowly at Arg (T3) to produce the characteristie biphasic pattern of inactivation. Localization of these splits was determined by sequencing N-termini of fragments after isolation on high resolution gel filtration columns [107]. 

Figure 2.4. Peptide fingerprinting by MALDI-TOF mass Spectrometry. Proteins are extracted and separated on by 2D gel electrophoresis. A spot of interest is excised from the gel, digested with trypsin, and ionized by MALDI. The precise mass of proteolytic fragments is determined by time-of- flight mass spectrometry. The identity of the protein is determined by comparing the peptide masses with a list of peptide masses generated by a simulated digestion of all of the open reading frames of the organism.

Figure 3.2. Stable isotope labeling for quantifying differential protein expression. Cell populations are grown in either 14N or 15N containing medium. Protein lysates are fractionated and separated by 2D gel electrophoresis. Protein spots are excised, digested with trypsin and the mass of the resulting peptides is determined by mass spectrometry. The presence of 15N results in a shift and creates two peaks for each peptide. The ratio of intensities of the peaks is indicative of the relative expression levels of the proteins. Spot A contains a protein that is expressed at similar levels in both cell pools. Spot B contains a protein that is expressed at higher levels in cell pool 2. Figure adapted from Oda et al. (1999).

Lindley, H. (1956) A new synthetic substrate for trypsin and its application to the determination of the amino acid sequence of proteins. Nature (London) 178, 647. 

The method of Kato and Nakai (27) for determining protein surface hydrophobicity was adapted for evaluating procyanidin binding to BSA and Gl. The procedure is based on the fact that the fluorescence quantum yield of cis-parinaric acid increases 40-fold when cis-parinaric acid enters a hydrophobic environment from a hydrophilic environment. The digestion of BSA by trypsin in the presence of procyanidin dimer, procyanidin trimer and black bean procyanidin polymer was evaluated by discontinuous sodium dodecyl sulfate (SDS) slab gel electrophoresis and a picryl sulfonic acid (TNBS) assay (28). 

A family of 100 hybridoma antibodies can typically provide 20 tight binders and these need to be assayed for catalysis. At this stage in the production of an abzyme, the benefit of a sensitive, direct screen for product formation comes into its own. Following identification of a successful catalyst, the antibody is usually recloned to ensure purity and stabilization of the clone, then protein is produced in larger amount (—10 mg) and used for determination of the kinetics and mechanism of the catalysed process by classical biochemistry. Digestion of such protein with trypsin or papain provides fragment antibodies, Fabs, that contain only the attenuated upper limbs of the intact IgG (Fig. 1). It is these components that have been crystallized, in some... 

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