Search

Using automated stepwise Edman degradation of suitable overlapping peptides, the complete amino acid sequence of the 268 residue peptida α1-CB7 from calf skin collagen was determined. The preparation and ordering of the chymotryptic, tryptic and thermolytic peptides is described in the preceding paper. As shown previously for other peptides from the helical region of collagen, glycine appears in every third position and proline is present in high amount. Hydroxylation of proline to 4-hydroxyproline and lysine to hydroxylysine only occurred in the Y position of the tripeptide unit Gly-X-Y. Non-random distribution of other amino acids between the X and Y positions was also observed. [ABSTRACT FROM AUTHOR]

The complete sequence (153 amino acids) of horse heart myoglobin has been established. The sequence of a peptide isolated from chymotrypsin hydrolyzates of globin allowed to fill a gap in the tentative sequence previously proposed; this peptide was studied by means of pepsin hydrolysis, action of N-bromosuccinimide on histidyl bonds, and Edman degradation method associated with dansylation. After hydrolysis of globin by chymotrypsin treated by TLCK, the same major peptides were found as after hydrolysis by non-treated chymotrypsin. These peptides were identified by ionexchange resins and bidimensional paper chromatography, and also by the determination of their amino-acid composition. The suppression of secondary cuts explains the presence of new peptides, whose the composition and N-terminal Sequences were determined, it was then possible to confirm several sequences which could be previously deduced from the study of split products after cyanogen bromide treatment. The specificity of chymotrypsin towards certain types of peptide bonds is discussed. Horse myoglobin, as compared with sperm whale myoglobin, contains 18 differences: 17 substitutions and one inversion. The major part of the substitutions are located ih N- and C-terminal sequences: they are all punctiform, resulting from the replacement of only one base in each codon. [ABSTRACT FROM AUTHOR]

The C-terminal fragment of hog pepsin, which had been obtained in the preceding study on the cyanogen bromide hydrolysate of this protein, was subjected to sequential studies. The 37-residue peptide was digested in independent experiments by trypsin, chymotrypsin, and pepsin, and the amino acid sequence of the arising peptides was determined. In parallel experiments the sequence of seventeen amino acid residues in the N-terminal region of the peptide was established by the Edman degradation technique. The obtained data permit the C-terminal peptide of hog pepsin to be ascribed the amino acid sequence Asp-Val-Pro-Thr-Ser-Ser-Gly-GluLeu-Trp-Ile-Leu-Gly-Asp-Val-Phe-Ile-Arg-Gln-Tyr-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-LysVal-Gly-Leu-Ala-Pro-Val-Ala. These results extend and pauly correct our previous knowledge of this region of the pepsin molecule as recorded in literature. The problem of the tryptophan content of pepsin is briefly discussed. [ABSTRACT FROM AUTHOR]

The reaction rates between dansyl chloride and water, amino acids or peptides are studied as a function of pH and temperature. The rate of hydrolysis of dansyl chloride is constant and low up to pH 9.5 and above this pH it increases rapidly. The various reactive groups of amino acids and peptides react with dansyl chloride in their unprotonated form. It is shown that a compromise for optima conditions of dansylation (pH and temperature) may be found, taking into account the rate of hydrolysis and the pk of the group to be dansylated. A complete chromatographie separation on Silica gel G thin layer plates of dansyl amino acids is proposed using 4 solvents. The quantitative recovery of the separated derivatives is described. The study of the acid hydrolysis of dansylated proteins shows that the release of the dansyl derivatives from the peptide chain is faster than that of free amino acids; it also shows that the destruction of dansyl amino acids occurs rather rapidiy. Therefore a hydrolysis time of 4 hours (110°) is suggested, except in the special cases of the release of dansyl valine, dansyl leucine or dansyl isoleucine which needs 18 hours of hydrolysis. For quantitative estimation of each dansyl derivative a correction factor is given, taking into account the loss during hydrolysis, the recovery from the plates and the individual fluorescence of any dansyl derivative as compared to the fluorescence of a reference compound. The general conditions described in this work require 1 nmole of material for qualitative determination of the N-terminal amino acids, and 5-10 nmoles for their quantitative estimation. Some examples (α-chymotrypsin, trypsinogen, ribonuclease, lysozyme aspartokinase and triose phosphate dehydrogenase) illustrate the efficiency of the method. [ABSTRACT FROM AUTHOR]

1. The replacement of the phenolic proton of tyrosine by alkyl, aryl or acyl groups completely abolishes the chymotryptic hydrolysis of tyrosine esters. Similarly, chymotrypsin fails to hydrolyze the tyrosyl bonds of dinitrophenyladrenocorticotropin. 2. Several model substrates have been synthesized and characterized. The action of chymotrypsin was followed potentiometrically in buffer containing some alcohol. The hydrolysis of the analogous compounds unsubstituted at the phenolic group was normal. 3. We conclude that inhibition is due, not to an inductive effect, but to a blocking effect which is markedly affected by replacement of the phenolic proton, even with a group as small as a methyl group. [ABSTRACT FROM AUTHOR]

It is shown that the change in activity and structure of acetyl-δ-chymotrypsin in the pH 7-11 range is not correlated with the ionizations of tyrosine residues. The pKs of the two titratable tyrosines of acetyl-δ-chymotrypsin are found to be 10.2 and 11.5, at 15° (9.6 and 10.8 after correction for electrostatic interactions), and those of the starting acetylated zymogen are 10.5 and 12.1 (10.0 and 11.5 after identical corrections). The plot of log rate of deacetylation of the titratable tyrosines versus pH is linear between pH 9 and pH 12. At a given pH there is a linear relation between the log deacetylation rate and the pKs of the residues, The pH rate profile of acylation of acetyl-δ-chymotrypsin by 4-carboxy-3-nitrophenyl- N,N-diphenylearbamate is bell-shaped, with a maximum at pH 7.9 at 15°, in 0.14 M KCl. The right side of the curve shows a pKapp of 8.9, whether the exposed titrable tyrosines of the enzyme are acetylated or not. A similar PKapp (9.1) is found for the pH-dependent change in optical rotation of the acetylated enzyme, when the exposed tyrosines are acetated. After correction for electrostatic interactions, this apparent pK becomes 8.4 and can be assigned only to the α-aminogroup of the N-terminal isoleucine. [ABSTRACT FROM AUTHOR]