Composition chymotrypsin

The influence of adsorption on the structure of a -chymotrypsin is shown in Fig. 10, where the circular dichroism (CD) spectrum of the protein in solution is compared with that of the protein adsorbed on Teflon and silica. Because of absorbance in the far UV by the aromatic styrene, it is impossible to obtain reliable CD spectra of proteins adsorbed on PS and PS- (EO)8. The CD spectrum of a protein reflects its composition of secondary structural elements (a -helices, / -sheets). The spectrum of dissolved a-chymotrypsin is indicative of a low content of or-helices and a high content of //-sheets. After adsorption at the silica surface, the CD spectrum is shifted, but the shift is much more pronounced when the protein was adsorbed at the Teflon surface. The shifts are in opposite directions for the hydrophobic and hydrophilic surfaces, respectively. The spectrum of the protein on the hydrophilic surface of silica indicates a decrease in ordered secondary structure, i.e., the polypeptide chain in the protein has an increased random structure and, hence, a larger conformational entropy. Adsorption on the hydrophobic Teflon surface induces the formation of ordered structural elements, notably an increase in the content of O -helices (cfi, the discussion in Sect. 3.1.4). 

Hydrolysis of peptides or proteins with acid yields a mixture of free a-amino acids. When completely hydrolyzed, each type of protein yields a characteristic proportion or mixture of the different amino acids. The 20 common amino acids almost never occur in equal amounts in a protein. Some amino acids may occur only once or not at all in a given type of protein others may occur in large numbers. Table 3-3 shows the composition of the amino acid mixtures obtained on complete hydrolysis of bovine cytochrome c and chymotrypsinogen, the inactive precursor of the digestive enzyme chymotrypsin. These two proteins, with very different functions, also differ significantly in the relative numbers of each kind of amino acid they contain. 

Partial hydrolysis of the peptide P using chymotrypsin as catalyst gave three peptides, X, Y, and Z. These were not sequenced, but their amino-acid composition was determined ... 

This information can be used to decide which of the alternative structures deduced above is correct. Chymotrypsin cleaves the peptide on the carboxyl side of the phenylalanine and tyrosine units. Only peptide M contains Phe, and if we compare M with the compositions of X, Y, and Z, we see that only X and Y overlap with M. Peptide Z contains the only Ala unit and must be the C-terminus. If we put together these pieces to get a peptide, P (which differs from P by not having the nitrogen corresponding to the ammonia formed on complete hydrolysis) then P must have the structure X-Y-Z ... 

Exercise 25-19 Eledoisin is a peptide isolated from the salivary glands of eledone, a Mediterranean eight-armed cephalopod. The peptide is a powerful hypotensive agent. Deduce a possible structure from the following information (1) Complete hydrolysis gives equal amounts of ammonia, Ala, Asp, Glu, Gly, lie, Leu, Lys, Met, Phe, Pro, and Ser. (2) No free amino /V-terminal group or free carboxyl C-terminal group can be detected. (3) Chymotrypsin hydrolysis forms two peptides, L and M. Their compositions are... 

From a rare fungus you have isolated an octapeptide that prevents baldness, and you wish to determine the peptide sequence. The amino acid composition is Lys(2), Asp, Tyr, Phe, Gly, Ser, Ala. Reaction of the intact peptide with FDNB yields DNP-alanine plus 2 moles of e-DNP-lysine on acid hydrolysis. Cleavage with trypsin yields peptides the compositions of which are (Lys, Ala, Ser) and (Gly, Phe, Lys), plus a dipeptide. Reaction with chymotrypsin releases free aspartic acid, a tetrapeptide with the composition (Lys, Ser, Phe, Ala), and a tripeptide the composition of which, following acid hydrolysis, is (Gly, Lys, Tyr). What is the sequence ... 

In Equation 11.13, A = k2k3[E]0[S]0/(k2 + k3) / [S]0 + k3Ks/(k2 + k3), which has the form of a Michaelis-Menten equation, B = [E]0[S]0 /(fe + 3) 2/([S]0+ Km(apparent)), and b is a composite rate constant describing the build-up of the acyl enzme intermediate (or, in the general case, the covalently bound enzyme intermediate). The non-linear plot of [Lg ] against time is shown in Fig. 11.10A for a typical substrate of a-chymotrypsin extrapolation of the linear portion gives the intercept shown which allows evaluation of B. 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

The effect of added co-solvent on the initial state is also important in more complicated reactions. For example, in the a-chymotrypsin-catalysed hydrolysis of p-nitrophenyl acetate and of N-acetyl-L-tryptophan methyl ester, the difference in the pattern of rates of hydrolysis when the solvent composition is varied can be attributed to the variation in the properties of the initial states of the esters (Bell et al., 1974). 

Figure 26-4. Principle of the Pancreolauryl and the NBT-PABA (V-benzoyl-i.-tyrosyl-P-aminobenzoic acid, bentiromide) test. The composite molecule consisting of a substrate and a marker molecule cannot be absorbed but can be cleaved intraduodenally by pancreatic enzymes (cholesterol esterase and chymotrypsin, respectively), leading to release of an absorbable marker substance. Following absorption and hepatic conjugation, the marker is excreted in urine. In pancreatic-insufficient patients, decreased secretion of pancreatic enzymes results in incomplete cleavage of the composite molecule. This results in decreased absorption and subsequent excretion of the marker, which can be measured photometrically.

In a chemical enzyme-modification experiment conducted by E. F. Jansen and colleagues in 1949, chymotrypsin was incubated with 32P-Iabeled DFP and then hydrolyzed with a strong acid. Separation of the constituent amino acids revealed 1 mol of labeled Ophosphorylserine per 25,000 g of chymotrypsin. Since DFP is a potent inhibitor of the enzyme chymotrypsin, what might we infer about the amino acid side-chain composition of the active site ... 

Figure 2. Predicted protein partition coefficient versus PEG molecular weight for lysozyme, chymotrypsin, albumin and catalase. Dextran molecular weight is 23,000 and polymer composition is PEG 6% Dx 8%.

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