Protein Fragmentation Chymotrypsin

Protein fragmentation, on the other hand, may be needed for functional activity of some proteins, such as chymotrypsin and insulin, which assume active forms after removal of amino-acid sequences in chy-motrypsinogen and proinsuhn. Additional complexity in analytical methodologies to deduce protein function in situ could also arise from a single protein exhibiting more than one function. Conversely, a given function may require integration of multiple proteins, or that many other proteins can perform the same function. 

To establish the amino acid sequence unequivocally it is necessary to have peptides with overlapping sequences. This may be accomplished by determining the sequence of fragments obtained from treating a second aliquot of the protein with chymotrypsin. If these fragments are then treated with trypsin as a check, peptides identical to those obtained previously by successive treatment with trypsin and chymotrypsin are obtained. Other proteolytic enzymes, such as pepsin, subtilisin, and papain, with wider specificity than trypsin and chymotrypsin have proved useful in sequencing of some proteins. 

Figure 37.2. Catalysis by the enzyme chymotrypsin of the cleavage of one peptide bond in a protein a proposed mechanism. Histidine and pro-tonated histidine act as general base and acid in two successive nucleophilic substitution reactions (a) cleavage of protein with formation of acyl enzyme and liberation of one protein fragment (6) hydrolysis of acyl enzyme with regeneration of the enzyme and liberation of the other protein fragment.

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]. 

A method for analyzing protein structure based on limited proteolysis. This method is especially useful in investigations of membrane proteins whose membrane association limits the repertoire of techniques that can be gainfully applied to infer structural features. For example, Davis et al used four proteases to assess the topology of yeast H -ATPase reconstituted into phosphatidyl-serine vesicles. Limited proteolysis by trypsin and a-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of... 

Because of its specificity for basic residues, trypsin converts a protein into a relatively small number of tryptic peptides which may be separated and characterized. Trypsin acts primarily on denatura ted proteins, and to obtain good results the disulfide bridges must be broken first. Chymotrypsin is less specific than trypsin and pepsin is even less specific (Table 3-2). Nevertheless, they can be used to cut a peptide chain into smaller fragments whose sequences can be determined. To establish the complete amino acid sequence... 

Fig. 2. The use of overlapping fragments to determine the sequence of a peptide. The protein is first digested with trypsin and the resulting peptides separated and sequenced. The protein is separately digested with chymotrypsin and the resulting peptides again separated and sequenced. The order of the peptide fragments in the protein can be determined by comparing the sequences obtained.

Analyze peptide fragments (PeptideMass of ExPASy) produced by treating a protein with the following amino acid sequence with chymotrypsin, proteinase K, and trypsin,. Deduce the specificities of these enzymes. 

Very fast electron transfers from P+ to bacteriochlorophyl (Bchl) and from (Bchl)- to QA do not depend on media dynamics and occur via conformationally non-equilibrium states (Fig.3.18). The dual fluorophore-nitroxide molecules (D-A) are also convenient objects for analysing the activity-dynamics relationship. The marked irreversible photoreduction of the nitroxide fragment of the dual probe incorporated into the binding site of HSA only took place when the nanosecond dynamical processes around the probe traced by ESR and fluorescence methods were detected (Rubtsova et al., 1993, Fogel et al, 1994 Likhtenshtein, 1986 Lozinsky et al., 2002). Similar results were reported for another model protein system, i.e. a-chymotrypsin with spin labeled methionin-92 groups (Belonogova et al., 1997). In the latter enzyme, the excited tryptophan group serves as an electron donor. 

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