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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

A new approach of predicting structural classes of protein domain sequences is presented in this paper. Besides the amino acid composition, the composition of several dipeptides, tripeptides, tetrapeptides, pentapeptides and hexapeptides are taken into account based on the stepwise discriminant analysis. The result of jackknife test shows that this new approach can lead to higher predictive sensitivity and specificity for reduced sequence similarity datasets. Considering the dataset PDB40-B constructed by Brenner and colleagues, 75.2% protein domain sequences are correctly assigned in the jackknife test for the four structural classes: all-α, all-β, α/β and α + β, which is improved by 19.4% in jackknife test and 25.5% in resubstitution test, in contrast with the component-coupled algorithm using amino acid composition alone (AAC approach) for the same dataset. In the cross-validation test with dataset PDB40-J constructed by Park and colleagues, more than 80% predictive accuracy is obtained. Furthermore, for the dataset constructed by Chou and Maggiona, the accuracy of 100% and 99.7% can be easily achieved, respectively, in the resubstitution test and in the jackknife test merely taking the composition of dipeptides into account. Therefore, this new method provides an effective tool to extract valuable information from protein sequences, which can be used for the systematic analysis of small or medium size protein sequences. The computer programs used in this paper are available on request. [ABSTRACT FROM AUTHOR]

For the elucidation of the amino-acid sequence of the carboxyl-terminal region of soybean trypsin inhibitor (Kunitz), fragments C and D were digested with trypsin, and the resulting peptides were separated by ion-exchange chromatography on Dowex 50X2 or by gel filtration on Bio-Gel P-4. Further fractionation and purification of the peptides were performed by ion-exchange chromatography on Dowex 1X2, by gel filtration on Bio-Gel P-2 or by high-voltage paper electrophoresis at pH 1.9 and 3.6. Three peptides were obtained in pure form from fragment C and ten peptides from fragment D, and their amino-acid sequences were determined by the direct Edman method and by carboxy- peptidase digestion technique. Overlapping peptides necessary for the alignment of the tryptic peptides from fragment D were obtained from a chymotryptic hydrolysate of fragment D. Nine main peptides and rune minor peptides were obtained. The amino acid composition and the partial amino-acid sequence of the 18 chymotryptic peptides of fragment D made it possible to establish the amino-acid sequence of the carboxyl-terminal region of the inhibitor. The complete amino-acid sequence of soybean trypsin inhibitor (Kunitz) deduced here was compared with that of Bowman-Birk inhibitor, another well-known soybean proteinase inhibitor. [ABSTRACT FROM AUTHOR]