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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 purified glycoprotein with blood-group-A specificity, was digested with insoluble pronase to give a serologically active product enriched in peptide-bonded serine and threonine residues; this was degraded with 0.25 M sulphuric in glacial acetic acid for 24 h. The resultant N-acetylgalactosaminyl-peptide were hydrolysed with 0.2 M hydrocholoric acid for 24 h at 110 °C. fractionation of the products on Sephadex G-10, followed by preparative separation on amino-acid analyzer and paper electrophoresis at pH 1.9 yielded the following homogeneous oligopeptides; one tripeptide with the N-terminal sequence Thr-Thr-Ser, three tetrapeptides with the sequences, Thr-Ser-Thr-Ser, Thr-Pro-Thr-Ser and Pro-Thr-Thr-Ser, one pentapeptide with the sequence Thr-Pro-Thr-Ser, one hexapeptide with the sequence Pro-Thr-Thr-Thr-Pro-Ser and one heptapeptide with the sequence Ala-Pro-Thr-Thr-Ser-Gly-Ser. Galactosamine was present in several of these fragments and the hexapeptide and heptapeptide both contained sufficient of this sugar for all the serine and threonine residues to be glycosylated. The following five tetrapeptides sequences were deduced from mixtures of two peptides which differed by the amino-acid substituent at only one position in the chain; Pro-Thr-Ala-ser, Thr-Thr Pro-Ser, thr-Thr-Ala-Ser, Ser-Pro-Thr-Ser, and Ser-Ala-Thr-Ser. A tripeptide mixture which differed only in the nature of the third amino acid yielded the following three tripeptide sequences: Pro-Thr-Ser, Pro-thr-Ala. These peptides do not indicate a clearly defined amino-acid sequence surrounding the glycosylated hydroxy amino acids but establish that in the human blood-group-specific glycoproteins there is close packing of the threonine in the region of the peptide moiety carrying the carbohydrate chains. [ABSTRACT FROM AUTHOR]

Peptide studies on two acidic proteins, A1 and A2, from 50-S ribosomes of Escherichia coli indicate a closely related primary structure. 1. Amino acid compositions of the tryptic peptides of the two proteins are identical, with exception of the amino terminal peptide. 2. The N-terminal amino acid sequence of A2-protein is Ser-Ile-Thr-Lys, while that of A1-protein is N-acetyl-Ser-Ile-Thr-Lys. 3. The estimated number of 120 amino acid residues in each polypeptide chain is consistent with the physical molecular weight estimate. 4. In 50% of the polypeptide chains, in both A1- or A2-protein, a specific lysine residue is replaced by ε-N-monomethyl-lysine. [ABSTRACT FROM AUTHOR]

The globin of sheep heart myoglobin was first cleaved by cyanogen bromide and the resulting fragments were fractionated by gel filtration and preparative paper electrochromatography; each fragment was submitted to endopeptidase digestion and the complete amino acid sequence has been determined. The intact globin and the median cyanogen bromide peptide were analysed by the protein sequanator (Edman). This analysis confirmed the sequence of the first 46 N- terminal residues of the intact globin and established the sequence of the first 39 N-terminal residues of the median cyanogen bromide peptide. Between sheep and beef myoglobin six differences over 153 residues can be noticed; among the six amino acid replacements, two correspond to the exchange of two bases and four to the exchange of one base in the coding triplets. [ABSTRACT FROM AUTHOR]

The αA2 chain of bovine α-crystallin was fragmented by means of cyanogen bromide treatment and by tryptic, chymotryptic and thermolytic digestions. Twenty tryptic peptides were obtained from the S-aminoethylated αA2 chain, accounting together for the complete sequence. The direct Edman degradation and the dansyl-Edman technique were used to determine the sequences of the tryptic peptides. The order of the tryptic peptides was deduced from overlapping peptides obtained by cyanogen bromide treatment, tryptic digestion of the maleylated chain and chymotryptic and thermolytic digestion of the S-aminoethylated chain. The sequence of the αA2 chain comprises 173 residues and corresponds to a molecular weight of 19832. [ABSTRACT FROM AUTHOR]

The substrate specificity of the sorghum acid protease was elucidated using synthetic peptides and pancreatic ribonuclease A as substrates. These studies revealed that the enzyme has a rather narrow and novel specificity requirement. The protease specifically cleaves the peptide linkages involving the α-carboxyl group of either aspartic acid or glutamic acid with the release of the acyl portion of these acidic amino acids. Further, the side chain carboxyl groups of aspartic acid arid glutamic acid in the peptides should be unsubstituted for the enzyme to act on the peptide, and therefore, glutaminyl and asparaginyl peptides are not cleaved by the enzyme. Studies carried out with performate oxidized ribonuclease and ribonuclease methyl esters have further substantiated the results obtained with the synthetic substrates. These results suggest that the sorghum acid protease will be very valuable in sequence analyses of proteins. [ABSTRACT FROM AUTHOR]

The study of the structure of the peptides arising from native, oxidized or reduced and maleinized bovine growth hormone on incubation with trypsin, ehymotrypsin and pepsin is reported. The data obtained permitted the assembly of a unique sequence of amino acids for the poly-peptide chain of the protein. Various corrections and one addition to the sequence previously communicated are made. [ABSTRACT FROM AUTHOR]

In order to ascertain the physiological role of the peptides which enhanced, the growth of Streptococcus thermophilus in milk, fourteen stimulating peptides were isolated and purified and their qualitative amino-acid composition was determined. The purification process was the following: after hydrolysis at pH 7.5 of whole casein by the neutral protease from Micrococcus caseolyticus the stimulating peptides were fractionated by gel chromatography (Bio-Gel P6), by column chromatography on cation-exchange resins AG 50W X2 (0.05 M pyridine-formate buffer at pit 3.10 to 1.5 M pyridine-acetate buffer at pH 5:10). Further purification was made by column chromatography on anion-exchange resins AG1 X2 (0.1 M pyridine-acetate buffer of pH 7.5 to 6 M acetic acid with a concave gradient system) and by paper chromatography (solvent: butanol-acetic acid-water, 4:1:5, v/v/v) and high tension electrophoresis (30 min; 60 V/cm). Gel filtration of enzymic digest of the whole casein led to six peptide fractions. Three fractions were stimulatory and exhibited a mean molecular weight of (B) 2500, (C) 1500, (D) 1000. Fraction C was the most stimulatory. Three other fractions were inhibitory and contained the highest molecular-weight peptides (about 5000) and peptides with high contents of aromatic residues. Ten and twenty peptide fractions were isolated respectively from Fractions B and D by chromatography on ion-exchange resins; but the peptides obtained after further purification were no more stimulatory. Eleven stimulatory and two inhibitory fractions were separated from Fraction C by column chromatography on cation-exchange resins. Purified stimulatory peptides were obtained from two fractions (C1 and C42) only, after anion-exchange resins (three peptides from Fraction C42, eleven peptides from Fraction C1). These fourteen purified stimulatory peptides display a very variable qualitative amino-acid composition. The polar part (Lys, Glx, Asx) was as important as the hydrophobic part (Pro, Leu, Val, Ala, Gly). The number of distinct amino-acid residues from one peptide was also quite variable: fourteen to four residues. Only lysyl residue was present in all peptides and tryptophan was not present. Three peptides that contained cysteine were issued from %-casein [18]; two peptides (C1a2α and C1C3) could be located respectively to Ala26…Gln22… and Ile26… Arg119 regions from bovine αs1B casein. A complete mixture of amino acids exerted growth stimulation. The set of the amino acids present in a stimulatory peptide was always stimulatory. These data substantiate the following conclusions: (a) stimulatory peptides acted as amino acid source; the amino acids have to be supplied in specific proportions in order not to disturb the cellular amino acids pool and the regulation of amino acid synthesis by Str. thermophilus; (b) the requirements in the peptide-length residues and lysyl residue could be explained by a control system of peptide intake, for example, by a peptide-transport system that would differ from that described in Escherichia coli. [ABSTRACT FROM AUTHOR]

Bence-Jones protein DEL was isolated from the urine of a patient with multiple myeloma by ammonium sulfate precipitation. Ion-exchange chromatography provided no further purification of the protein; the ammonium sulfate precipitate consisted of only one protein moiety as tested by polyacrylamide electrophoresis. Protein DEL contains 213 amino acids and has a molecular weight of 22500. Eighteen peptides have been isolated by ion-exchange chromatography after tryptic digestion of the completely reduced and aminoethylated protein. Sequence studies were performed mainly on these tryptic peptides. In addition, 14 chymotryptic pepfides were isolated and characterized to obtain overlapping peptides for the whole V-region and for some tryptic peptides of the C-region. Protein DEL is related to subgroups III and IV of the γ-chains as indicated by a deletion of three amino acids after position 27, a common pattern of these two subgroups. Furthermore, position one is deleted in protein DEL as in the other proteins of subgroups IH and IV but in contrast to subgroups I and II of the γ-chains. However, marked differences exist between protein DEL and the proteins of subgroups IH and IV. a) The deletion of two amino acids in positions 95 and 96, characteristic of subgroup IV, was not found in protein DEL. b) 19 of the 27 linked exchanges specific for subgroup IV are substituted in protein DEL. Although the insertion of two amino acids after position 94 is the same as in subgroup III, a high exchange rate was observed for proteins DEL and Sh. Therefore it is concluded that protein DEL does not belong to either subgroups III or IV, but is the first example of a new subgroup of the γ-chains. [ABSTRACT FROM AUTHOR]

Describes reaction of glutamate dehydrogenase with glyoxal and NaB[sup 3]H[sub 4] and the labelled protein digested with trypsin. Isolation and identification of radio peptides in the primary structure of the enzyme; Small amounts of radioactivity contained in many peptides; Unspecific reactions with glyoxal; Identification of lysine-27 as the residue reacting with glyoxal; Conclusion that lysine residues.

1. Tryptic hydrolysates of denaturated glutamic-aspartic transaminase fractionated on a Sephadex G-25 column, gave an elution diagram containing six main fractions. When peptide maps were developed from the transaminase digest 37 spots were detected on the paper sheet. 2. It was demonstrated that the transaminase fractions (groups of peptides) as well as some of the individual peptides eluted from the map spots, possessed immunological activity. This was proved by theft capacity to interact with rabbit pig heart antienzyme and rabbit antiserum against the pig-heart whole tryptic transaminase hydrolysate as well as to inhibit the homologous transaminase-antitransaminase reaction. Higher immunological reactivity was found among peptides moving faster in electrophoresis and chromatography. 3. The catalytic activity of rabbit transaminases (heart, liver, skeletal muscles), was inhibited to a substantial degree by the rabbit anti-pig heart transaminase. The above enzymes however. as was otherwise expected, gave negative precipitin reactions with antitransaminase. A comparative study of the reactivity between transaminases from pig, ox and rabbit and rabbit antitransaminases against pig and ox heart enzymes as well as against pig-heart whole tryptic transaminase hydrolysate is presented here. [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]

Tryptic peptide maps of human liver alcohol dehydrogenase show this protein to be homologous to the corresponding protein of the horse. A subunit molecular weight very close to 40000 is established for the human enzyme. Sequence analysis of about one quarter of the tryptic peptides of human alcohol dehydrogenase show identical residues to the horse enzyme at about 90% of the positions, which is considered fairly representative of the general resemblance between the two proteins. All amino acid exchanges found are compatible with one-base mutations in the genetic code. Two different types of subunits of human liver alcohol dehydrogenase are identified. They are essentially similar but differ at some positions, one of which is No 43 (valine in one subunit and alanine in the other). Still other subunit types may exist. The known occurrence of isoenzymes of human liver alcohol dehydrogenase may therefore be explained at least in part by subunits of different primary structures. The amino acid differences between the subunits of the human enzyme are not found at the same positions as those between the two types of subunit of the horse enzyme. The structural differences between subunits from the two species seem greater than between the subunits within either species. Isoenzyme differences may, therefore, have evolved independently in the two species. The suggestion that some of the amino acid exchanges between the horse subunits may be directly involved in the substrate binding is supported. The isoenzyme and species differences at position 43 is only three residues away from the reactive "active site" cysteine residue, The region around this important residue is thus not kept constant in liver alcohol dehydrogenase during evolution. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of neurotoxin I of the North African scorpion Androctonus australis Hector collected in Algeria (area of Chellala) is described. S-Methylation of cysteine residues formed by reduction of disulphide bonds was found useful for their identification in sequence determination using the phenylisothiocyanate method. From the N-terminal sequence (38 residues) established in the protein sequenator, and from the sequence of chymotryptic peptides the complete sequence of the protein has been deduced. The molecule consists of a single polypeptide chain cross-linked by four disulphide bridges. An isoneurotoxin (I') has been found in the venom of A. australis collected in Tunisia (area of Tozeur). It differs from neurotoxin I in the replacement, of the valine residue in position 17 by an isoleucine residue. [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]

A new procedure for the determination of the N-terminal groups in peptides and proteins is described. The free amino-groups are allowed to react with 2-fluoro-3-nitropyridine or 2-fluoro- 5-nitropyridine in slightly alkaline solution, the resulting derivatives are easily hydrolyzed in dilute hydrochloric acid in sealed tubes to give the corresponding nitropyridyl amino acids. Most of these compounds are quantitatively recovered under these conditions; in particular it is possible to estimate proline and glycine as N-terminal residues. After isolation the nitropyridyl amino acids can be estimated spectrophotometrically; in some cases it is possible to regenerate the parent amino acid by treating the derivatives with strongly alkaline solutions. The adequacy of the present techniques in relation to those of other workers was tested on insulin, lysozyme and aldolase; the advantages and peculiarities in comparison with these procedures are discussed. [ABSTRACT FROM AUTHOR]

The λ-chains of normal pig γG-immunoglobulin were digested with pepsin in 1 M formic acid. Cystine-containing peptides were isolated by gel filtration on Sephadex G-25 and by chromatography on Dowex 50 and Dowex 1. The following structures were found:[this equation cannot be presented in ASCII text] In addition to these peptides obtained in a high yield similar peptides were found but their complete structures were not determined in view of their low yield. The structures found in the λ-chains display a high degree of similarity with corresponding structures known to exist in human Bence­Jones proteins of the L-type. [ABSTRACT FROM AUTHOR]

The main polypeptide chain of the lens protein α-crystallin (αA2) is N-terminally acetylated and contains two methionine residues. The chain is initiated by the incorporation of a methionine residue exclusively donated by methionyl-tRNAfMet. This N-terminal methionine is not removed during polypeptide chain synthesis. The internal methionine residue is donated exclusively by one of the methionyl-tRNAMet species. When lens messenger RNA coding for the αA2 chain is translated m a reticulocyte cell-free system, the newly synthesized polypeptide chain carries an acetylated methionine in the N-terminal position. [ABSTRACT FROM AUTHOR]

The complete amino acid sequence of neurotoxin II of the North African scorpion Androctonus auatratis Hector has been determined. From the N-terminal sequence (43 residues) obtained by automatic phenylisothiocyanate degradation in the protein sequenator, and from the sequence of tryptic peptides obtained by digestion of the reduced and methylated toxin of which amino groups had been previously masked by reaction with ethylthioltrifluoroacetate the complete amino acid sequence of the protein has been inferred. It is composed of a single polypeptide chain of 64 amino acid residues, including 8 half-cystines, ended by an histidinamide residue at the C-terminus. [ABSTRACT FROM AUTHOR]

The suppressor-sensitive mutants of phage ø29,susB47 and susN212, do not induce the synthesis of proteins NP1 and H, respectively, when they infect Bacillus subtilis su-. A new polypeptide of molecular weight 75000, smaller than that of protein NP1, is produced in susB47-infected cells. An analysis of the methionine-containing tryptic peptides of protein NP1 and the 75000-molecular- weight polypeptide showed that both proteins are related. When mutants susB47 and susN212 infect B. subtilis su+3, a small amount of proteins NP1 and II, respectively, is synthesized, the level of the suppression being about 10%. The results indicate that the B. subtilis su+3 strain is a suppressor of nonsense mutations. [ABSTRACT FROM AUTHOR]

Histone F3 has been fragmented into 10 non-overlapping fragments. With the aid of the automatic Edman degradation procedure for proteins using 3-(N,N-dimethylamino)propyne buffer the first 48 amino-acid residues in the uncleared protein have been positioned. The remainder of the amino-acid sequence of this protein was elucidated by subjecting the aligned peptides to the automatic degradation. The amino acids obtained during the degradation were quantitated and identified by gas chromatography and amino-acid analysis. The primary structure of this histone is compared to the corresponding fraction from calf which had been independently determined by another laboratory. [ABSTRACT FROM AUTHOR]

The homology between the 30-S ribosomal protein S1 and interference factor i has been investigated. The two proteins are indistinguishable by gel electrophoresis under various conditions. Similar peptides are obtained by cleavage with cyanogen bromide. After labelling with N-ethylmaleimide, enzymatic digestion yields homologous labelled peptides. This homology is confirmed by immunological data. Characteristic activities of the two proteins have been compared. Both bind polyuridylic acid and stimulate polypeptide synthesis on this template by S1-depleted ribosomes. Furthermore, S1 inhibits the translation of the coat protein cistron on MS2 RNA, while it has little effect on the overall translation of T4 mRNA, two characteristic properties of interference factor i. S1 and factor i are thus physically and functionally indistinguishable, which suggests that they have identical poly-peptide chains. Implications are discussed in terms of function.

Histidine decarboxylase from Micrococcus sp.n. was carboxymethylated and two types of subunits, I and II, were separated in excellent yield by exclusion chromatography on Sephadex G-100 in 8 M urea. Total compositions, number of tryptic peptides and N-terminal groups of the subunits were analyzed. The larger subunit I is composed of all amino acids commonly found in proteins and has a blocked N-terminal residue, whereas the smaller subunit II does not contain proline, histidine or cysteine and has N-terminal methionine. The number of tryptic peptides detected in peptide mapping experiments of subunit I is about 27 and that of subunit II is about 15. These figures are in good agreement with the total number of lysine and arginine residues determined by amino acid analysis, if subunit I contains about 270 residues, corresponding to a molecular weight of 29000, and subunit II about 70 residues, corresponding to a molecular weight of 7000. It is concluded that histidine decarboxylase contains two apparently homogeneous but completely different polypeptide chains and that the native enzyme of molecular weight 110000 is a hexamer with three subunits of each type. [ABSTRACT FROM AUTHOR]

The article reports on different science experiments made by scientists regarding the creation of life through proteins. The first science experiment was made in 1955 by Stanley Miller wherein the discovery of proteins was founded from the condensation of amino acids. Another experiment was done by Clifford N. Matthews and by Robert E. Moser which challenged the discovery of Miller due to its conclusion that proteins appeared without the formation of amino acids. It was supported by laboratory investigations at Monsanto because it showed that peptides with twelve amino acids were formed from hydrogen cyanide and water with ammonia.

A synthetic multichain polypeptide, (T,G,)-A--L 509, was trace labelled with 125I in the tyrosine end groups, composing the main antigenic determinants, and used to study the distribution of antigen in the draining lymph nodes after injection into the footpads of mice. The polypeptide was administered: (a) in saline solution into unprimed mice—in which form it is not demonstrably immunogenie; (b) in Freund's adjuvant into unprimed mice—in which form it is immunogenie; and (c) in saline solution to primed mice, so as to give a booster response. Experiments comparable to (c) were also done with [125I]haemocyanin. At various time intervals from 12 hours to 21 days later sections of the draining nodes were examined by radioautography and methyl green—pyronine staining, or by a combination of immunofluorescent staining for antibody containing cells with radioautography. In unprimed mice, irrespective of whether they made antibody, labelling was found predominantly in tile phagocytic cells of the medulla and the cortical sinus, but definite weaker labelling was also seen in the germinal centres. The label persisted throughout the period of observation, but tended to become more prominent with time in the germinal centres of mice making antibody m response to antigen m Freund's adjuvant. In primed mice the label was rapidly and predominantly concentrated in germinal centres, where it persisted throughout the period of observation while its intensity gradually diminished in other sites. The distribution of label in germinal centres was in a lace-like or dendritic pattern, similar to that described by Nossal and co-workers, and did not correspond to the lymphocytes or pyroninophilic cells in the centres. Grain counts were made over specific antibody containing cells in both primary, and booster responses to (T,G)-A--L. Such cells sometimes lay close to and sometimes many cell diameters distant from phagocytic cells containing concentrations of the antigen, but the cells themselves did not have grain counts significantly different from the background count. If the antigen remained undegraded, under the conditions of the experiment it would have been possible to detect the presence of fifteen molecules or less in a cell. Reasons are given for supposing that 125I constituted a valid label of the main antigenic determinant groups, of which there were a maximum of 100 per molecule. [ABSTRACT FROM AUTHOR]

The protein α-crystallin from calf eye lenses can be isolated in very high yield by zonal centrifugation. No other purification steps are required in order to obtain preparations the purity criteria of which allow sequence studies after dissociation into polypeptide chains, By this method evidence is provided that α-crystallin is a population of aggregates built up by four different polypeptide chains, occurring in an almost, constant ratio independent on the size of the aggregate. [ABSTRACT FROM AUTHOR]

The crystal protein of Bacillus thuringiensis var. tolworthi has been separated into two polypeptide chains having molecular weights of 55000 (A) and 120000 (B). The ratio of A to B in the crystal appears to be 2:1 on a molar basis. Differences in amino-acid composition and biological activity exclude the possibility that the larger polypeptide chain is a dimer of the smaller one. The full toxicity of the unfractionated protein to larvae of Pieris brassicae is retained by fraction A, whereas fraction B is non-toxic. [ABSTRACT FROM AUTHOR]

The determination of the amino acid sequence of the C-peptide of human proinsulin is presented. Two constituent fragments of C-peptide, Chy 1+ 2 and Chy 3 (residues 1-24 amd 25-31, respectively) were liberated by mild chymotryptic digestion and isolated gel filtration and ion exchange chromatography. From Chy 1 + 2 was obtained seven smaller peptides by further digestion with chymotrypsin. The sequence analysis of these fragments allowed an overall structure to be assigned. Papain digestion of the intact C-peptide provided 12 peptides, the compositions of which provided unequivocal confirmation for the proposed sequence. [ABSTRACT FROM AUTHOR]

The monomer molecular weight of C-phycocyanin from three different algal sources was investigated. The techniques used for determining molecular weight included high speed sedimentation equilibrium, osmotic pressure measurements in 6 M guanidine hydrochloride, and cyanogen bromide peptide analysis. All measurements were consistent with a monomer molecular weight of 30000 ± 1000 for C-phycocyanin isolated from all algal sources. [ABSTRACT FROM AUTHOR]

The recent sequence determination of histone F2b has allowed a detailed nuclear magnetic resonance study of the conformational changes and interactions of this protein with (a) increasing ionic strength, (b) increasing concentration and (c) change of pH. In all cases it has been found that a specific region of the polypeptide chain is involved in the induced changes: with increasing ionic strength the changes and interaction can be located in the segment of the polypeptide chain from residues 60 to 102; part of this segment is also involved in the specific aggregation that occurs on increasing the concentrations, while for the pH induced changes the same segment together with additional residues at each end is involved in the observed changes. [ABSTRACT FROM AUTHOR]

Large peptides containing the allotypic determinants can be isolated from tryptic digests of rabbit IgG reduced with dithiothreitol. Their antigenic activity is detected by simple screening for interference in the allotype anti-allotype reaction. Active peptides are obtained for A1 A2 and A3 allotypes and carry apparently the major determinants involved in the allotype anti-allotype reaction. Further characterization of these peptides is in progress. [ABSTRACT FROM AUTHOR]

Molecular fragments of various lengths arc formed by treating acid-soluble collagen with collagenase from Clostridium histolyticum. Fragments 78, 105 and 125 nm long can be isolated by fractional ammonium sulphate precipitation. Their position in the collagen molecule has been determined by electron optical investigations. Denaturation of the individual fragments yielded peptides of the α1- and α2-chains which could be separated by CM-cellulose chromatography. These peptides, α1 (78) and α1 (105), are in the region of two of those obtained by cyanogen bromide cleavage, namely, α1-CB7 and CB6; α1 (125) is superimposed on α1-CB3, CB7 and CB6. In order to obtain smaller fragments of these CB peptides of the α1-chain, the three, fragments from collagenase treatment were cleaved further with cyanogen bromide and the resultant pepfides were isolated and characterized. Their position along the α1-chain was determined electron microscopically after renaturation to the triple helical structure. Each fragment of the α1-chain yields two chromatographically and electrophoretically distinguishable peptides which have been designated α1 and α1*. The difference between the two was found to reside in the—COOH end of the molecule: the α1* peptide contains seven amino acids more than the α1. [ABSTRACT FROM AUTHOR]

Penicilloylated proteins which are found as an impurity in 6-aminopenicillanic acid can be exhaustively digested by pronase to yield amino acids and small peptides. This degradation converts the point polyvalent antigens into a mixture of mostly monovalent haptens which are much less immunogenic and less capable of eliciting immune reactions in sensitized animals. The degradation of the protein impurity can be carded out in 6-aminopenicillanic acid preparations, since even a large excess of 6-aminopenicillanic acid does not appreciably inactivate or inhibit the enzyme. In order to avoid the contamination of 6-aminopenicillanic acid with a proteolytic enzyme, pronase was converted into a water insoluble form by coupling it with bromoacetyl cellulose or carboxymethyl Sephadex. These insoluble derivatives of pronase were found to retain their activity and broad specificity. Thus, they can be readily removed from the medium upon completion of the impurity degradation, to be used repeatedly in a continuous process. [ABSTRACT FROM AUTHOR]

The determination of tryptophan and cysteine content in proteins by means of the selective reaction of sulfenyl halides with these amino acids has been carefully investigated. By reaction with 2- or 4-nitrophenylsulfenyl chloride in aqueous acetic or formic acid, the nitrophenyl chromophore is covalently bound to the tryptophan and the cysteine residues of the protein, giving a thioether and a mixed disulfide respectively. The cysteine content has been determined spectrophotometrically at 450 nm from the 4-nitrothiophenol released from the mixed disulfides of the cysteine residues after exposure of the 4-nitrophenylsulfenyl chloride-labelled protein in 0.1 N NaOH (at this wavelength the corresponding chromophore covalently bound to tryptophan does not absorb). Tryptophan has been estimated in the sulfenylated proteins by spectrophotometric determinations at 365 or 328 nm of the 2- and 4-nitrophenylsulfenyl chromophores (εM 4000 and 11700 respectively), following removal of the yellow label from the cysteine residues, by dissolving the modified protein in 0.1 N NaOH. The procedure has been shown to be accurate for both amino acids with a wide variety of proteins. [ABSTRACT FROM AUTHOR]

The hemoglobins of a child homozygous for the Leporeboston gene have been analyzed. Only hemoglobin F and Lepore were observed. Hemoglobin Lepore was isolated and the peptide chains of this hemoglobin were separated. The amino acid composition of the Lepore chain and of the tryptic peptides obtained from this chain was determined. The peptides isolated accounted for the amino acid composition of the Lepore chain and indicated that this chain is 146 amino acid long. The switch region from the δ-like sequence of the Lepore chain to the β-like sequence has been localized between residue 87 and residue 116. [ABSTRACT FROM AUTHOR]

Glucagon is a polypeptide of 29 residues, which can be crystallised. Optical rotatory dispersion in dilute neutral, acid and alkaline solution suggests that the poypeptide chain is largely random, but contains about one turn of α-hehx, which is elfininated in 6 M guanidine hydrochloride. Using thermal difference spectra, nuclear magnetic resonance, and the temperature-dependence of the optical rotatory dispersion as criteria, it appears that glucagon possesses no defined tertiary structure in solution, but may be regarded as being in a state of αhelix⇄ random coil equilibrium, near the high-temperature end of the transition range. That there is no steric objection to the formation of a highly α-helical conformation is demonstrated by optical rotatory dispersion measurements in 2-chloroethanol solution, in which the protein becomes largely helical. A rough calculation indicates that in aqueous solution at pH 2 the transition mid-point is at about —40°. From changes in the proton magnetic resonance spectrum when glucagon is transferred from an aqueous environment into 6 M guanidine hydrochloride, it is deduced that the short helical segment in the chain in the aqueous state contains most of the long aliphatic side chains, and since the sequence is known it has been possible to identify such a segment uniquely near the C-terminal end. On standing in acid solution the viscosity increases and a birefringent gel is formed. Sedimentation studies indicate the formation of large aggregates. On further standing, or warming, a precipitate appears which has the appearance of long fibrils in the electron microscope. Infrared spectra of the gel, of solid films and of the precipitated material show that in all these states the glucagon is in the form of antiparallel β-chains. [ABSTRACT FROM AUTHOR]

Certain glutamyl-containing polypeptides react with homologous sheep antibody only in the presence of divalent cations, and are dissociated with chelating agents. Near ultraviolet circular dichroism and hydrogen exchange studies show reversible conformational changes occurring in the antibody during specific interaction, but only at low antigen-antibody ratios. [ABSTRACT FROM AUTHOR]

Bovine β-lactoglobulin-A was hydrolysed with trypsin to yield a mixture of peptides which would not precipitate with rabbit antibody against the native protein, but would still inhibit the antigen-antibody reaction. The hydro- lysate was fractionated by ion exchange chromatography and found to contain seven antigenically active fractions. Two of these fractions were found to be homogeneous peptides. Each inhibited the reaction of antigen with antibody against the intact protein in haemagglutination, flocculation and passive cutaneous anaphylaxis reaction in guinea-pigs. One of the peptides had a molecular weight of 950 and the other had a molecular weight of 575. Both contained about equal numbers of polar and apolar amino acids. [ABSTRACT FROM AUTHOR]

The purification and chemical characterization of three Bence-Jones proteins and one light chain of an IgG myeloma protein of the rat strain LOU/Wsl are described. The amino acid composition of the four chains is very similar but not identical. Fingerprint analyses show a pattern of peptides common to all four proteins as well as unique features. The compositions of the common peptides are given. The data indicate that the four light chains are of the kappa type. [ABSTRACT FROM AUTHOR]

Reduced, S-aminoethylated guinea-pig α-lactalbumin was cleaved with eyanogen bromide at its single methionyl residue. The products were separated gel by filtration and characterized by amino acid analysis and N-terminal group determination, as a 90-residue peptide from the N-terminal of the protein (N-terminal lysine) and a 33-residue peptide from the C-terminus of the protein (N-terminal aminnethyl cysteine). The amino acid sequences of both these peptides were determined by examination of the peptides obtained by digestion of the purified fragments with trypsin, ehymotrypain or thermotysin. The complete amino acid sequence of guinea-pig α-lactalbumin thus obtained is discussed in relation to the known sequences of other members of the Iysozyme-α-lactalbumin group. [ABSTRACT FROM AUTHOR]

By peptic or tryptic proteolysis of different fragments of bovine encephalitogenic protein, a set of small fragments covering the following regions of the native protein have been obtained: 44-68, 44-89, 75-91, 93-116, 114-130, 134-150, and 154-170 (the amino acid residues are numbered according to [1]). These fragments were used in the macrophage migration inhibition technique in guinea pigs (an assay which is vividly considered as a correlate in vitro to delayed type hypersensitivity) to study the localization of antigenic determinants in the protein. The results indicate the presence of at least one determinant in each of regions 44-68, 134-140. and 104-170. Furthermore, the rather strong determinant, which has previously been localized around the single tryptophan residue in the protein does not seem to be contained within peptide 114-130, as this peptide is considered unreactive in the assay. On the other hand, this peptide shows full encephalitogenic activity. [ABSTRACT FROM AUTHOR]