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1 A new diagonal electrophoretic 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 ɛ-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-Tyr-Thr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The three basic amino 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 sequences of the two proteins.

Amino acids are quantitatively recovered from aminoacyl-tRNA in filter paper strips by treatment with 0.3 m ammonium carbonate. A simple apparatus is described for deaminoacylation and separation of amino acids and tRNA. The amino acids, thus recovered, can be directly analyzed by paper chromatography since ammonium carbonate is removed by lyophilization. Nonvolatile reagents such as Tris-HCl and sodium carbonate also hydrolyze aminoacyl-tRNA, but unless the products of deaminoacylation are desalted, the presence of these reagents gives rise to chromatographic artifacts such as peak broadening, appearance of multiple peaks, displacement of R f values, etc. tRNA remaining after deaminoacylation retains only a small fraction of its original capacity to bind amino acids. Part of this loss of activity appears to be due to structural damage to tRNA in the course of washing and counting procedures and in part, due to the effect of filter paper in shifting the equilibrium of the reaction toward hydrolysis of aminoacyl ester bond.

A method is described for the separation and quantitative determination of the weakly basic (cross-linking) amino acids of elastin. A 6 N HCl hydrolyzate is submitted to high-voltage electrophoresis at pH 3.8. At least eight spots can be identified in the weakly basic region and quantitated by the ninhydrin-photodensitometric method. Some of these are spot 3 for desmosine + isodesmosine, and 7 for lysinonorleucine. Quantitative data given for two typical elastin preparations are in agreement with direct amino acid analysis. Preparative electrophoresis can also be carried out and used for the isolation of small quantities of the unknown spots separated by this procedure.

Die Organisatorregion von Huhnchenembryonen wurde mit CAP behandelt und die Wirkung des CAP auf schwefelhaltige Aminosauren papierchromatographisch untersucht. Die Analyse ergab, dass Glutathion starker beeinflusst wurde als Methionin. Die Reduktion des Glutathiongehaltes konnte dem Abfall der Induktionskapazitat des Hensenschen Knotens entsprechen, der durch die Behandlung mit CAP hervorgerufen wird.

SummaryN-substituted amino acid can be separated by paper chromatographic method. Also, N-substitution leads to the extinction of fluorescence of amino acids.

1. The peptides obtained by tryptic digestion of S-[14C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.

Extensive pronase digestion of the desialized bovine submaxillary major mucin (BSM) resulted in a mixture of glycopeptides. Separation of the glycopeptides into nine fractions, fractions a to i , was achieved by linear gradient elution at constant pH (2.6) of 0.05–0.2 m pyridine-formate buffer from Dowex 50-X2 (pyridinium form). Glycopeptide b 6 was isolated from fraction b by preparative high-voltage paper electrophoresis, and glycopeptides g 5 and e 7 from fractions g and e , respectively, by preparative high-voltage paper electrophoresis and preparative paper chromatography, in succession. Homogeneity of these glycopeptides was shown by high-voltage paper electrophoresis at pH 1.9, 3.7, and 6.5 and by paper chromatography. The results of the N-terminal analysis by dansylation method with or without sequential Edman degradation and the analytical data before and after a β-elimination reaction of glycopeptides b 6 , g 5 , and e 7 indicated the structures of these glycopeptides as follows: Glycopeptide b 6 , seryl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide g 5 , glycyl-( O -α- N -acetylgalactosaminyl-)-serine; glycopeptide e 7 , seryl-( O -α- N -acetylgalactosaminyl-)-threonyl-glycyl-( O -α- N -acetylgalactosaminyl-)-serine.

Peptides comprising the 7–11, 11–20, and 12–20 sequences of human encephalitogenic basic protein (HEP) were synthesized by the solid-phase procedure, purified by gel-filtration and high-voltage paper electrophoresis, and characterized by amino acid analysis, thin-layer chromatography, high-voltage paper electrophoresis, and paper chromatography.The peptides were studied for the capacity to induce E.A.E. when injected with Freund's complete adjuvant (FCA) into guinea pigs. The same peptides were also studied for the capacity to inhibit development of E.A.E. in guinea pigs injected with peptide/FCA 28 days before injection with encephalitogenic HEP in FCA. The results were as follows: (1) all three peptides induced mild clinical E.A.E. but the 12–20 peptide induced severe disease in five out of 40 guinea pigs; (2) none induced histological lesions typical of E.A.E.; (3) none induced antibody reactive with 125I-HEP; (4) none induced delayed sensitivity to the homologous peptide or HEP; (5) injection of the 12–20 peptide in FCA inhibited the development of histological E.A.E. in guinea pigs injected later with HEP/FCA, whereas the 7–11 and 11–20 sequences did not; (6) humoral immunity to injection of HEP/FCA was not prevented by prior injection of the synthetic peptides in FCA.

Aspartic acid was replaced in position 1 of angiotensin II (ATII) with several amino acids, to assess the possible influence of the N-terminal amino acid for (a) the intrinsic activity, (b) the affinity, and (c) the metabolic degradation of agonist analogues of ATII. Some of the substitutions in position 1 were used in combination with replacement of Phe by Gly or Leu in position 8, to obtain the corresponding antagonist.The compounds were tested in vivo (rat blood pressure) and in two in vitro preparations (rat stomach and rabbit aorta strips). The oil immersion technique, described by Kalsner and Nickerson (1968) (Can. J. Physiol. Pharmacol. 46, 719–730), was used to study the disposition of the peptides by vascular smooth muscles (rabbit aorta strips). Degradation of the peptides by purified aminopeptidases was evaluated in vitro by measuring the fragments on paper chromatography. Potency of antagonists was estimated in vivo (ID50) and in vitro (pA2 values): duration of action was established by infusing the inhibitors intravenously into anesthetized rats and testing the effect of standard doses of angiotensin before and after.The results indicate that replacement of Asp with other amino acids does not influence the intrinsic activity, but can either increase or decrease the affinity in vitro or the potency in vivo. 1-Sar-ATII, and 1-D-Ala-ATII are more potent and longer acting than 1-Asp-ATII on isolated intestinal and vascular smooth muscles, but not in vivo. On the contrary, 1-β-Asp-ATII and 1-β-D-Asp-ATII are more potent than 1-Asp-ATIIin vivo, but not on rabbit aorta strips. Rate of relaxation of rabbit aorta strips suspended in oil, after contraction with submaximal doses of several analogues of ATII, are significantly slower than relaxation after 1-Asn2-ATII and 1-Asp-ATII. A close parallelism between the diminution of the relaxation rate in oil and the degradation by aminopeptideses in vitro was observed, suggesting that metabolic degradation may be the major factor determining relaxation of rabbit aorta in oil after contraction with one of these peptides. Potencies of antagonists in vivo and in vitro are increased by replacing Asp with Sar. Substitution of Asp with β-Asp or β-D-Asp brings about a slight increase of potency in vivo but not in vitro. It appears that firm binding and prolonged occupation of receptors by sarcosyl derivatives are the primary factors contributing to increase the potency and to prolong the duration of action of antagonists, while prevention or reduction of metabolic breakdown by aminopeptidases is much less efficient.

SUMMARY The concentration and ratio of nonessential to essential amino acids were determined by paper chromatography in serum and erythro-cytes of fasting rats and in animals that received a non-protein diet, and compared with rats on a stock diet. Our results show that very dicfferent changes are brought about in the non essential to essential amino acid ratios, in the amino acid concentrations and in their relative erythro-cyte/plasma distribution by the different dietary treatments. The changes induced by the non-protein diet resemble those reported in clhildren suffering from protein-calorie malnutrition. The fact that the changes are already apparent after only 3 days of dietary treatment seem to indicate that abnormal ratios reflect more the quality of the diet than the nutritional status.

1. Triose phosphate isomerase was prepared by chromatography on DEAE-cellulose of an (NH4)2SO4 fraction of an extract of homogenized chicken breast muscle. The product is homogeneous on gel electrophoresis and is suitable for growing crystals for X-ray work. The specific activity is 10000 units/mg and the value for E0.1%280 is 1.20. 2. Comparison between the sum of the amino acid compositions of the tryptic peptides of the protein and the amino acid composition obtained on total hydrolysis of the protein indicates that the relative subunit mass is about 27000. 3. These data, together with the results of the examination of the amino acid compositions of a number of minor peptides, the number of peptides in the tryptic digest and the complete amino acid sequences of the tryptic peptides (the determination of which is described here), give no indication that the subunits are dissimilar. 4. A tentative amino acid sequence is presented for the protein, in which the ordering of the tryptic peptides is derived by homology with the sequence of the rabbit muscle enzyme (Corran & Waley, 1973). 5. An appendix describes the use that was made of mass spectrometry in the determination of some of the sequences. Mass-spectrometric data have been obtained for 35 residues, that is about 15% of the total sequence of the protein. 6. An extended version of the present paper has been deposited as Supplementary Publication SUP 50025 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

Synthetic luteinizing hormone-releasing hormone (LH-RH) lost both its immunore-activity and hormonal activity on incubation with hypothalamic or cerebrocortical slices or homogenates. This inactivation was shown to be due to degradation of the decapeptide by soluble enzyme(s) present in the 100,000 × g supernatant fraction of the homogenates. The supernatant derived from one rat hypothalamus was capable of destroying 1 μg of exogenous LH-RH within 5 min. The hexapeptide pGlu-His-Trp-Ser-Tyr-Gly was identified as the major radioactive breakdown product of [pGlu-3-3H] LH-RH, and tentative evidence for the formation of the tetrapeptide Leu-Arg-Pro-Gly-NH2 was obtained by sequential electrophoresis and paper chromatography. These findings suggest that the Gly-Leu bond may be the preferred site of cleavage.

1. 1. High-voltage paper electrophoretic and paper chromatographic separation of free amino acids in the pupa of Glossina palpalis gave a minimum of seventeen ninhydrin-positive spots. 2. 2. Most of these ninhydrin-reacting substances are neutral amino acids, and α-alanine has the highest concentration.

A method is described for isolation of insulin from chicken pancreas which makes use of modifications of established procedures to deal with the low insulin content of the tissue and permits isolation of significant quantities of the hormone. The insulin isolated is essentially homogeneous by paper chromatography and polyacrylamide gel electrophoresis. The amino acid composition of the insulin and of the separated A and B chains is consistent with the known amino acid sequence of chicken insulin. (Endocrinology 83: 1323, 1968)

Guinea-hen egg-white lysozyme was obtained in a chromatographically pure state by ion-exchange chromatography on Amberlite CG-50. The reduced alkylated enzyme was subjected to digestion with trypsin. The resulting peptides were purified by a combination of Dowex 1-X2 chromatography and electrophoresis and chromatography on paper. The amino acid sequence of these peptides was determined chiefly by the Edman dansylation procedure. Alignment of the tryptic peptides into a single chain containing 129 amino acids was determined from basic overlap peptides obtained by digestion of the enzyme with chymotrypsin. 10 replacements were noted between guinea hen and hen lysozymes. Further comparisons were achieved with 4 other bird egg-white lysozymes the structures of which were already known.

Certain strains of Streptococcus pyogenes, Type 12 elaborate a polypeptide which is nephritogenic in experimental animals. It appeared to be a pure, nonmigrating component by paper chromatography, to be electrophoretically pure when analyzed by the Spinco electrophoretic system, and to be homogeneous by ultracentrifugal analysis. Quantitative amino acid analyses of acid-hydrolyzed samples revealed 19 amino acids and trace amounts of five unidentified components. Glycine, proline, alanine, glutamic acid and aspartic acid accounted for 70% of the amino acids found. Wilzbach tritiated samples were injected intraperitoneally into 20-g C57B1 male mice. Radioautographic evidence revealed that a high percentage of the injected material concentrated in renal tissue.

Treatment of glycopeptides prepared from rabbit brain glycoproteins with NaOH or NaOH-NaBH 4 released approximately 10% of the carbohydrate as low molecular weight oligosaccharides. After treatment with alkali alone, a disaccharide and a trisaccharide were isolated by gel filtration and preparative paper chromatography, and have been identified as N- acetylneuraminyl -(2→6)-O-2- acetamido -2- deoxy - D - galactose (Component B-I) and di -N- acetylneuraminyl - D - galactose (Component B-III). The N -acetylgalactosamine in Component B-I was obtained as preformed Morgan-Elson chromogen, and Components B-I and B-III were not obtained when the glycopeptides were treated with alkaline NaBH 4 under conditions which prevent peeling. These results indicate that the trisaccharide (Component B-III) was originally present as a substituent at C-3 of the N -acetylgalactosamine involved in the carbohydrate-peptide linkage, and was eliminated as a result of alkali treatment. A disaccharide (Component M) was isolated after alkaline NaBH 4 treatment of the desialylated glycopeptides, and yielded equimolar amounts of galactose and galactosaminitol after hydrolysis. Smith degradation of Component M gave galactose and threosaminitol, indicating that the disaccharide was galactopyranosyl -(1→3)-O-2- acetamido -2- deoxy - D - galactitol . From the isolation of Components B-I, B-III and M after NaOH and NaOH-NaBH 4 treatment of the glycopeptides, it is concluded that the major alkali-labile oligosaccharide of brain glycoproteins is the pentasaccharide di -N- acetylneuraminyl - D - galactopyranosyl -(1→3) [N- acetylneuraminyl -(2→6)]-O-2- acetamido -2- deoxy - D - galactose .

The separate treatment of bovine trypsin and the Kunitz soybean trypsin inhibitor with phenylglyoxal resulted in the modification of both of the arginine residues of trypsin and an average of 5 of the 9 arginine residues of the trypsin inhibitor. When the trypsin-soybean trypsin inhibitor complex was modified under the same conditions, a total of 3 arginine residues had reacted, from which it was concluded that 4 arginine residues had been buried in the complex. Analysis of the trypsin and soybean trypsin inhibitor which were dissociated from the phenylglyoxalated complex revealed that one of the buried arginine residues was derived from trypsin and an average of 2.5 to 3 residues from the trypsin inhibitor. Peptide maps of digests from trypsin which had been phenylglyoxalated in the presence of soybean trypsin inhibitor and from trypsin alone showed one arginine-containing peptide common to both digests. This peptide was isolated by ion-exchange chromatography and two-dimensioned paper chromatography and electrophoresis. From its composition and the known sequence of bovine trypsin, this peptide was shown to contain arginine-55 which is thus presumed to be located at or near the zone of contact in the trypsin-soybean trypsin inhibitor complex.

1. When purified calf thymus histones were incubated with [1-14C]acetyl-CoA and treated to remove the acid-soluble fraction with trichloroacetic acid, a large amount of radioactivity still remained in the 15 % trichloroacetic acid-insoluble precipitate. 2. This incorporation of radioactivity into the protein did not seem to be enzymatic, since it took 60 min boiling of the histone to diminish only about 60 % of the incorporation. The boiled histones were eluted faster than the “native” histone on Sephadex G-50 column in 0.02 M HCl, suggesting a change in the tertiary structure of the proteins. Furthermore, polylysine was efficiently acetylated. 3. The reaction was dependent on pH, period of incubation, ionic strength and ionic species. The activation energy for the reaction was 7.5 kcal. 4. All of the various histones were acetylated, but slightly lysine-rich histone was acetylated the most. Among various other proteins and polypeptides tested, only polylysine and polyarginine were highly acetylated. 5. At maximum acetylation, one out of every 120 amino acid residues in histone was acetylated. Amino acid analysis of proteolytic enzyme digests of [acetyl-14C]histone and [acetyl-14C]polylysine revealed that practically all of the incorporated radioactivity was as e-N-acetyllysine. The presence of e-N-[acetyl-14C]acetyllysine was also confirmed by paper chromatography.

Deoxyribonucleoprotein content of human brain was 1.11 mg/g grey matter and 1.76 mg/g white matter; the DNA component was 0.50 mg/g grey matter and 0.72 mg/g white matter. The base composition of the DNAs is given. The amino acid composition of the conjugated histones was investigated quantitatively by paper chromatography and the basic constituents found to represent 25% total histone amino acids. A residual protein fraction was isolated from the nuclei and its amino acid pattern reported. Comparisons of these constituents were made with those in other human and animal tissue.