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Various workers have interpreted their data on the rates of alkaline denaturation of ferrihemoglobin either as a consequence of the presence of two distinct species or as a result of two consecutive first order steps involving an intermediate. Our results with a chromatographically purified homogeneous sample indicate that the rate of alkaline denaturation support the latter hypothesis. The rates of denaturation of ferrihemoglobins A, S and C under the same conditions differ, and it is concluded that the amino acid sequence of the ferrihemoprotein plays a significant role in the conformational stability of the molecule. [ABSTRACT FROM AUTHOR]

Histones from the fruit fly Ceratitis capitata have been obtained and characterized by amino acid analysis and polyacrylamide gel electrophoresis. It has been found that the five major histone groups are present in this insect. The fractionation of histones has been achieved by using acid or organic selective extractions. Ceratitis capitata F1 exhibited a reduced electrophoretic mobility when compared with the mammalian corresponding histone. On the contrary, fly F2A1 mobility was similar to that of mammalian homologous histone. Some differences were found, however, in amino acid composition. It has been found that cysteine does not occur in fly F2A1, but this amino acid seemed to be present in the F3 histone, which aggregated under oxidizing conditions. A strongly basic protein was isolated together with histones when pharate adult chromatin was used as starting material. Amino acid analysis of this protein revealed the presence of hydroxyproline (19%), as well as high amounts of threonine (30%) and arginine (23%). It was found that this specific protein was not a nuclear component and it seemed to be located at the pharate adult envelope; the name of purarium-specific protein is proposed for this protein. Its strongly basic nature is judged to be the cause by which it became attached to chromatin during the isolation procedure. [ABSTRACT FROM AUTHOR]

Reaction of RNAase A with bromoacetate at pH 5.5 for 1–42 days results in multiple reactions. Alkylation of residues proceeds in the sequence: (1) N-1 of histidine-119; (2) methionine (probably methionine-30); (3) N-3 of histidine-12; (4) N-3 of histidine-105 and N-3 of 1-carboxymethyl histidine-119; and (5) lysine-1. Both histidine-12 and histidine-119 of the same active site are carboxymethylated. A derivative carboxymethylated at both active site histidines is obtained in 1 day and probably some of this derivative is obtained in short reaction times. This is contrary to the conclusions of earlier investigations. Histidine-48 undergoes little or no reaction. The results are in accord with the X-ray structure of RNAase. [ABSTRACT FROM AUTHOR]

1. Evidence is presented that an iodinating treatment of cytochrome c, in Tris buffer, pH 8.6, 8M urea, leads to a chemical modification of the thioether bridges which link the heme to the polypeptide chain It is suggested that the thioether sulfur atoms are oxidized to the sulfoxide stage. At low iodine levels (6-8 moles of iodine per mole of cytochrome c) no other amino acids are affected, as shown by ammo acid analysis and use of 125I. 2. The main piece of evidence for the transformation is that treatment of tins presumed cytochrome c 14,17-disulfoxide Ieads to heme cleavage. 3. Both the oxidation and the cleavage reaction are dependent upon conformational factors. 4. A possible mechanism is proposed for the cleavage reaction which led to the prediction, borne out by experiment, that cleavage can also be effected by acetic anhydride. [ABSTRACT FROM AUTHOR]

After reduction and carboxymethylation, crystallized cytochrome b2 has been separated by gel filtration into two main fractions and a minor one The latter is probably an aggregate The apparent molecular weight of the major fractions has been determined by acrylamide gel electrophoresis m the presence of 0.1% sodium dodecyl sulfate, and found to be of the order of 36000 and 21000. Their amino acid composition is very different. Their sum gives a composition in good agreement with the composition corresponding to the minimum molecular weight of unfractionated cytochrome b2. On the basis of the evidence presented in the paper and of the known molecular weight of crystallized cytochrome b2, it is suggested that the protein is an oligomer of the type (αβ)4. [ABSTRACT FROM AUTHOR]

From the tryptic hydrolyzate of pig immunoglobulin &kappa-chains 6 cystine-containing fragments were isolated. Amino acids analysis showed that they contained 37-70 amino acid residues and were of a variable nature. There exists a linear relationship between the specific electrical charge of the fragment and the ionic strength of the buffer by which the fragment is eluted from a column of QAE-Sephadex. Fragments, the disulfide bond of which was split by oxidative sulfitolysis, were split by thermolysin and the hydrolyzates were compared by the method of peptide maps. All the maps of fragments tested contained the peptide Ile-ThrSerCysSSO3-Arg (where CysSSO3 = S-sulfo-cysteine) which represents the vicinity of the half-cystine 24 of pig κ-chains, as shown by us previously. The nature of the peptide maps indicates that the individual fragments originate in the same section of the primary structure of the N-terminal half of the κ-chains. Each of the isolated fragments was a mixture of variants with an identical specific charge but with some amino acid replacements. [ABSTRACT FROM AUTHOR]

Whole histone and five fractions were prepared from the sperm of tile sea urchin Arbacia lixula. They were characterized by starch gel electrophoresis and amino acid analyses. The fractions are classified as follows: ϕ1(very lysine-rich), ϕ2b(lysine-rich), ϕ2a2 (intermediate type), and ϕ2a1 and ϕ3 (lysine-poor). They have many similarities with calf thymus histone fractions, F1, F2b, F2a2, F2a1 and F3, in this order. Nevertheless, differences exist, the most important being a higher arginine content in several fractions, and the high proportion of the very lysine-rich fraction found in the sperm of A. lixula. [ABSTRACT FROM AUTHOR]

Calf corneas were extracted with a buffer containing M CaCl2, 0.05 M Tris and 0.02 M citric acid, at pH 7.5. The residual stroma was digested successively by colagenase and pronase. The glycopeptides obtained were separated by gel filtration and high voltage electrophoresis. The sugar and amino acid content of the isolated glycopeptides was determined. Two types of sugar chains were detected: (a) chains with a more complicated structure, containing galactose, glucose, mannose and glucosamine; (b) disaccharidic and monosaccharidic side chains containing galactose and glucose or galactose alone and linked O-glycosidically to the OH groups of hydroxylysine. The Hyl-Gal-Glc compoud was isolated and identified with the compound isolated by Butler and Cunningham from calf skin collagen, by the study of its composition, thin layer chromatography and high voltage electrophoresis. The stroma of the calf cornea contains about 4 times more hexoses in the small glycopeptides of type b (di- and monosaccharides) than in the higher molecular weight mannose-containing chains. [ABSTRACT FROM AUTHOR]

Amino acid analyses on thioredoxin reductase indicate the following amino acid composition: Asp65Thr43Ser26Glu60Pro17Gly63Ala58Val32Met12Ileu39Leu53Tyr16Phe18Try2Lys23His18Arg29 (total half-cys)8 (CONH2)59. The enzyme consists of two identical or very similar polypeptide chains joined by noncovalent bonds. This is indicated from experiments employing ultracentrifugation in urea or guanidine hydrochloride, from the analysis of tryptic peptide maps and from quantitative determinations of NH2-terminal amino acids. Carboxymethylation of the enzyme with iodoacetic acid under different conditions showed that each polypeptide chain of the native enzyme contains one disulfide and two free sulfhydryl groups, the latter buried in the enzyme structure. Titration of the enzyme with NADPH under anaerobic conditions demonstrated that the complete reduction of one molecule of enzyme requires four molecules of NADPH. The results indicate that the enzyme contains two redox acceptors in addition to the two molecules of flavin adenine dinucleotide. Evidence is presented that these redox acceptors are identical with the two disulfides of the enzyme. Spectral studies of thioredoxin reductase employing anaerobic titration with substrate did not reveal the formation of a stable red 2-electron intermediate similar to that observed for two other flavoproteins, lipoyl dehydrogenase and glutathione reductase. This finding suggests that the catalytic mechanism for thioredoxin reductase is different from that of lipoyl dehydrogenase and glutathione reductase. [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]

A method for amino acid analysis by double isotope labeling has been developed using ⊃3H-labelled dansyl chloride, and 14C-labelled amino acids as internal standards. As little as 20 pmol of an amino acid can be accurately measured by this method. The amino acid compositions of several peptides, determined using about 50pmol peptide, are reported and compared with the results obtained using conventional high-sensitivity ion-exchange chromatography. The correspondence is very good. The amino acid composition of a tryptic peptide containing the active site disulphide bridge of the lipoamide dehydrogenase component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli has been shown to be identical to that of the corresponding peptide from the 2-oxoglutarate dehydrogenase complex of the same organism. This result is consistent with the view that the lipoamide dehydrogenase components of both complexes have a common amino acid sequence. [ABSTRACT FROM AUTHOR]

Chloroform-soluble proteins have been isolated free from phosphatides from rat liver mitochondria, microsomes and cytosol by chromatography on Sephadex LH-20 columns. The amino acid composition of the chloroform-soluble and insoluble proteins has been estimated. The chloroform-soluble proteins from mitochondria and cytosol have a higher content of hydrophobic and a lower content of acidic amino acids compared to the corresponding chloroform-insoluble proteins. No marked differences were found between chloroform-soluble and insoluble proteins from microsomes. In contrast to the acidic chloroform-insoluble proteins, all chloroform-soluble proteins are basic. From the amino acid composition the amount of chloroform-soluble proteins has been estimated. Mitochondrial membranes contain 1.6%, mitochondrial matrix proteins less than 0.025%, microsomes 3.0% and the cytosol 0.15% from total proteins. The molecular weight distribution of chloroform-soluble proteins synthesized in mitochondria has been measured by gelelectrophoresis. The molecular weights range from about 7000 to 30000. The turnover of chloroform-soluble proteins from mitochondria and cytosol is higher than from the corresponding chloroform-insoluble proteins. A part of mitochondrial chloroform-soluble proteins (about 30%) is synthesized within the mitochondria. [ABSTRACT FROM AUTHOR]

Hexokinase has been purified from pig heart to a specific activity of 80 units/mg. The enzyme has an s20 of 5.11 ± 0.15S and a molecular weight of 97000. Dodecyl-sulphate-polyacrylamide electrophoresis and maleylation of the enzyme each suggest that it contains a single polypeptide chain. Amino-acid analysis reveals similarities between the compositions of heart and brain hexokinases. Steady-state kinetic investigations at sub-optimal substrate concentrations are consistent with a mechanism in which at least one of the substrates is in equilibrium with its enzyme · substrate complex. [ABSTRACT FROM AUTHOR]

Antigenic components were solubilized from washed rat thymocyte membranes by reaction with citraconic anhydride. The product remained soluble after partial decitraconylation. The weight composition of the soluble material was 60% protein, 10% RNA, 80% DNA, 20% carbohydrates and 9% phospholipids. The antigenic activity of the soluble material defined as the capacity to neutralise the cytotoxic activity of an antithymocyte serum, is not affected by exposure to temperatures up to 45 °C, by exposure to pH values ranging from 3 to 10, and by removal of the nucleic acid components. Proteolytic digestion with trypsin brought about a 30% loss of antigenic activity, while digestion with papain abolished all the antigenic activity. Periodate oxidation destroyed 65% of the antigenic activity. Reduction of the oxidized mixture with sodium borohydride restored part of the lost activity. The oxidized and reduced mixture contained 50% of the original antigenic activity. Treatment with chaotropic agents only slightly affected the antigenic activity of the solubilized material. However, when mercaptoethanol was added to the chaotropic agents, most of the antigenic activity was lost. Exposure of the solubilized material to organic solvents resulted in partial loss of antigenic activity. Thus, it seems that most of the antigenic determinants are conformation-dependent. Gel filtration of nuclease-treated, solubilized membrane preparation on columns of Sepharose 6 Beluted with Tris-saline buffer, fractionated the antigenic material into 3 main components. Amino acid analysis showed that each of the components contained about 17% basic, 23% acidic anb 31% hydrophobic amino acid residues. Significant differences were observed in the carbohydrate and lipid contents of these peaks. The material contained in the high molecular weight component, eluting near the void volume, had a specific activity up to 10-times higher than that of the unfractionated mixture. Electrophoresis on polyacrylamide gels containing sodium dodecylsulfate and mercaptoethanol revealed that a fully citraconylated preparation (which is antigenically inactive) contained the same electrophoretic components as those present in a decitraconylated, antigenically active, preparation. The fractions obtained by gel filtration on Sepharose 6B contained components of various molecular weight. The high molecular weight peak (which contained most of the antigenic activity) displayed one major band with a molecular weight of about 70000. In the absence of reducing agents bands of higher molecular weight were observed. The data suggest that peak I contained aggregated material. Indeed, upon gel filtration on Sepharose 4B eluted with 1% sodium dodecylsulphate, two fractions of a lower molecular weight were obtained. The larger molecular weight component on dodecylsulfate polyacrylamide gel electrophoresis displayed one major band of molecular weight of 70000, while the smaller molecular weight component displayed one major electrophoretic band of molecular weight of about 60000, and an unresolved mixture of smaller molecular weight components, some of which may be phospholipid. Antisera elicited by the purified antigenic components were active against thymocytes in the cytotoxic assay, and were able to prolong skin graft survival in an allogeneic system. [ABSTRACT FROM AUTHOR]

The alkaline protease from Acremonium kiliense Grütz which is sensitive to düsopropyl-phosphofluoridate, can be crystallized from solutions of ammonium sulphate. The amino-acid analysis of the protein shows no special features and the N-terminal residue is alanine. The Molecular weight was found to be 27000 by sedimentation-equlibrium in the ultracentrifuge, and between 28500 and 31000 by electrophoresis on polyacrylamide gels in the presence of sodium dodecylsulphate. The enzyme is highly resistant to inactivation by 8M urea in which it is fully active. It is inactivated by heating to above 55 °C, and this temperature effect is unchanged in urea. The enzyme is irreversibly inactivated by 6M guanidine hydrochloride, Sodium dodecylsulphate at 0.1% and 1.0% (w/v) inactivates the enzyme only very slowly at pH values above 7: but at lower pH the inactivation is very rapid. The circular dichroism spectra of lysozyme, serum albumin and chymotrypsinogen dissolved in dodecylsuphate and 2-mercaptothanol at pH 7.1 are very similar at low wavelengths ad suggest that they all have a significant proportion of α-helical structure. The spectrum of the Acremonium enzyme under the same conditions is quite different and shows no features suggesting a particular structure. These observations suggest that any complex formed between the enzyme an dodecylsuphate may not have the same structure as of he dodecylsulphate protein complexes. The enzyme has three disulphide bonds per molecule. Only two of them are available for reaction in 8 M urea, but the third is exposed in 6 M guanidine hydrochloride. [ABSTRACT FROM AUTHOR]

Collagen was isolated from the skins of normal and lathyritic rats. Amino acid analysis, sugar analysis and analysis of the subunit composition indicated that the two types of collagen differed only with regard to the degree of cross-linking. Investigations by physical chemical methods showed that the lathyritic collagen preparations behaved as free monomers below 15 °C at physiological ionic conditions with the following characteristics: s200,w = 2.92 S, D200,w = 0.78 × 10-7cm²/sec, [n] = 11.1 dl/g. Mr from light scattering, sedimentation equilibrium and sedimentation-diffusion was 310000–320000. Light scattering data agreed with a rod-like structure and the molecular length from light scattering, frictional ratio and intrinsic viscosity was 300 nm. Preparations of normal collagen were aggregated under physiological ionic conditions and different reasons for the aggregation are discussed. It is suggested that lathyritie collagen should be a good model substance in studies at the molecular level of fibrfllogenesis and interactions between collagen and other macromolecules. [ABSTRACT FROM AUTHOR]

The complete amino-acid sequence of cytochrome c from Ginkgo biloba L. (Maidenhair tree) has been determined on 0.5 µmol protein; chymotryptic and tryptic peptides were analysed by the dansyl-Edman method. The molecule consists of a single polypeptide chain of 113 amino-acid residues and is homologous with the other plant cytochrome-c sequences which have been determined [1]. The additional residues present as compared to other plant cytochromes c are at the C. terminus of the protein. Residue 59 was glycine; previously all mitochondrial cytochromes c have invariant alanine in this position. Two residues of the unusual amino acid ε-N-trimethyl-lysine are present in the protein. [ABSTRACT FROM AUTHOR]

Treatment of the highly aggregated S-carboxymethyl derivative of the α-protein of epidermis with trypsin or chymotrypsin results in progressive hydrolysis to small peptides with no helical structure. However, experiments done at 4°C at enzyme-to-protein ratios of 1/1000 for short periods of time resulted in the formation of three distinct components of varying size. X-ray diffraction and amino acid analyses of protein from the components indicate that the α-protein breaks down into fragments of helical and non-helical structure. [ABSTRACT FROM AUTHOR]

This article presents a study to determine the estimation of protein-bound iodine in plasma or serum. The estimation of protein-bound iodine in plasma or serum is, at present, the nearest to direct assay of the circulating thyroid hormone. It is thus surprising that very little has been reported on the protein-bound iodine levels in phenylketonuria. The seventeen phenylketonurics were aged 14-55 years. The diagnosis in each was confirmed by amino-acid analysis. All were in good health and no drugs were administered either during or immediately prior to the investigation.

This article comments on the studies of a phosphorylated derivative of 3-amino-1,2,4-triazole (3AT) formed by the action of yeast hexokinase. Two methods were used to isolate phosphorylated 3AT from reaction mixtures in which commercial yeast hexokinase preparations had acted on 3AT. The results show that phosporus was detected only after acid hydrolysis, and which had certain properties in common with the glucose derivative of 3AT. Infra-red studies of this new compound revealed several differences in absorption bands which served to establish it as a separate compound derived from the amine glucoside by phosphrylation.

Lysozyme from human parotid saliva was purified to the point where it was judged, by the criteria of ultracentrifugation and disk electrophoresis, to be homogeneous. The parotid lysozyme had a specific activity 3.5 times greater than that of hen egg-white lysozyme and a composition that was different, especially in regard to tyrosine, serine, and glutamic acid content. The molecular weight was determined to be around 14,300. When tryptophan residues were modified with N-bromosuccinamide, no lytic activity was found, which indicates that the tryptophan residues may play an important role in the maintenance of the parotid lysozyme activity. [ABSTRACT FROM AUTHOR]

The article examines the amino acid composition of the insoluble material of decalcified dentin. Research methods include the demineralization of dentin in unerupted third molars with a solution of ethylenediaminetetraacetic acid or EDTA at pH 6.5. The insoluble protein is classified as a collagen with a high content of glycine, proline, and hydroxyproline.

The article presents research on the variations in the amino acid composition of dentin and enamel. Tooth tissues were separated and decalcified by Battistone's and Burnett's methods. The tissues of powdered teeth were separated by a modified flotation technique or the enamel was separated from the dentin by chipping. The test results are also provided.

The article presents the results of a study which investigated the chemical structure of the non-decayed segments of teeth containing dental caries. An overview of related previous research is provided, along with details of the experimental protocol. It was found that the amino acid content of proteins which were derived from the enamel of carious teeth was significantly different from that of healthy teeth.

Delipidation by ethanol-diethyl ether at -10°C of human serum high-density lipoprotein (HDL, d 1.063-1.21) or of its subclasses HDL2 (d 1.063-1.120) and HDL3 (d 1.120-1.21), yielded proteins-αP, αP2, and αP3-containing 3% phospholipid (largely lecithin) and 3.3% carbohydrate (glucosamine: l-fucose: d-galactose, d-mannose: sialic acid, 1.00:41:0.56:0.31).Solubility data and analytical ultracentrifugal analyses indicated that, upon lipid removal, HDL protein aggregates readily; the aggregation is dependent upon pH and ionic strength of the solvent medium. Subunits of 21,000 mol wt were obtained by acetylation or addition of sodium dodecyl sulfate (SDS).HDL and αP elicited in the rabbit a similar immunological response. By agar gel immunoelectrophoresis both anti-HDL and anti-αP sera detected a major and two minor antigenic determinants in HDL, HDL3, αP, αp2, and αP3. HDL2, antigenically homogeneous, gave an immunoelectrophoretic pattern of HDL3 upon mixing with αP. αP, αP2 and αP3 exhibited a single antigenic determinant after treatment with SDS (0.5 m) or upon acetylation.Native or delipidated forms of HDL, HDL2, and HDL3 were separated by vertical starch gel electrophoresis into several components, which showed identical reactions against anti-HDL or anti-αP sera.The data suggest that (a) the proteins of HDL, HDL2, and HDL3 are made of subunits, probably identical, of an average molecular weight of 21,000; (b) the difference in antigenic behavior between HDL2 and HDL3 is due to the presence in the latter of a lipid-poor protein; (c) antigenic polymorphism of αP is probably related to the presence in solution of monomeric and polymeric forms having different reactivity against anti-HDL and anti-αP sera.