scholarly journals Selective purification of the thiol peptides of myosin

1968 ◽  
Vol 107 (4) ◽  
pp. 531-548 ◽  
Author(s):  
A G Weeds ◽  
B S Hartley
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Acid Oxidation ◽  
Thiol Groups ◽  
Peptide Maps ◽  
Thiol Peptides

1. A method for selective purification of thiol peptides is described. Thiol groups in a protein are treated with radioactive cystine by disulphide–thiol interchange. The labelled cystine peptides in a digest can then be fractionated for peptide ‘maps’. Performic acid oxidation of paper strips containing the radioactive peptides followed by further ionophoresis yields the purified cysteic acid peptides. 2. The thiol peptides in a peptic digest of cystine-exchanged myosin were purified in this way, and their amino acid sequences were determined. 3. The conclusion that myosin contains at least 16, and probably between 20 and 22, unique thiol sequences indicates that the molecule consists of two chemically equivalent components.

scholarly journals Tamm–Horsfall urinary glycoprotein. The chemical composition

1970 ◽  
Vol 120 (2) ◽  
pp. 417-424 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acid ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Amino Acid Residues ◽  
Carbohydrate Composition ◽  
Thiol Groups ◽  
Terminal Amino Acid ◽  
Cystine Content ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. A revised amino acid and carbohydrate composition of human Tamm–Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm–Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11–12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4°C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate–protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.

scholarly journals The amino acid sequence of cysteic acid-containing peptides from performic acid-oxidized ovotransferrin

1970 ◽  
Vol 116 (3) ◽  
pp. 515-532 ◽  
Author(s):  
T. C. Elleman ◽  
J. Williams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Cystine Content

1. The half-cystine content of ovotransferrin, measured as cysteic acid, was 31mol/80000g of protein. 2. The amino acid sequences of cysteic acid-containing peptides from performic acid-oxidized ovotransferrin were studied. 3. 34 unique cysteic acid residues were identified. 4. It is concluded that hen ovotransferrin does not consist of two identical halves or subunits.

scholarly journals Thiol and disulphide contents of hen ovalbumin. C-Terminal sequence and location of disulphide bond

1970 ◽  
Vol 116 (4) ◽  
pp. 555-561 ◽  
Author(s):  
L. A. Fothergill ◽  
J. E. Fothergill
Keyword(s):  
Acetic Acid ◽  
Iodoacetic Acid ◽  
Disulphide Bond ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Acid Oxidation ◽  
Double Labelling ◽  
Thiol Groups ◽  
Terminal Sequence

1. The thiol and disulphide contents of hen ovalbumin were investigated by p-chloromercuribenzoate titration, by determination of cysteic acid content after performic acid oxidation, by measurement of uptake of radioactive iodoacetic acid, and by assay of S-aminoethylcysteine after reaction with ethyleneimine. All results showed that ovalbumin had 6 half-cystine residues. Experiments with and without reducing agents demonstrated that there were 4 thiol groups and 1 disulphide bond. 2. A peptide containing equimolar amounts of S-carboxymethyl-cysteine, serine, valine and proline, but no lysine or arginine, was obtained by radioactive labelling of the cysteine residues with iodo[14C]acetic acid followed by electrophoretic and chromatographic separation of tryptic digests. It was concluded that the C-terminal sequence of ovalbumin is -Cys-Val-Ser-Pro. 3. The location of the disulphide bond was studied by using a double-labelling technique. It was shown that one end of the disulphide was located in this C-terminal peptide.

Amino Acid Sequence Studies of Horseradish Peroxidase. I. Tryptic Peptides

1972 ◽  
Vol 50 (1) ◽  
pp. 44-62 ◽  
Author(s):  
K. G. Welinder ◽  
L. B. Smillie ◽  
G. R. Schonbaum
Keyword(s):  
Amino Acid ◽  
Horseradish Peroxidase ◽  
Amino Acid Sequence ◽  
Paper Electrophoresis ◽  
Iodoacetic Acid ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Acid Oxidation ◽  
Tryptic Peptides ◽  
Tryptic Digest

Commercially available horseradish peroxidase (RZ 3.1) was characterized with respect to its homogeneity by (1) chromatography on CM-cellulose, (2) disc gel electrophoresis in alkaline and acidic buffers, (3) micro-scale sucrose gradient isoelectrofocusing in pH 3–10 and pH 8–10 gradients, (4) gel isoelectrofocusing in pH 3–10 and pH 8–10 gradients, and (5) amino acid and hexosamine analyses. The preparation was found to be highly homogeneous except by pH 8–10 gel isoelectrofocusing which resolved it into several very close bands. This heterogeneity has been assumed to reflect differences in carbohydrate composition rather than in the amino acid sequence or composition. Amino acid analyses after performic acid oxidation yielded eight cysteic acid residues per mole of enzyme. Since no S-carboxymethylcysteine was recovered after treatment of the protein in 8 M urea with iodoacetic acid, it was concluded that the enzyme has four disulfide bridges. Peptides resulting from a tryptic digest of the heme-free enzyme were purified by high-voltage paper electrophoresis and subjected to sequence analysis. Several half-cystine sequences were elucidated after isolation of the radioactive peptides from a tryptic digest of the reduced and 14C-S-carboxymethylated protein. The complete sequences of 21 and partial sequences of three tryptic peptides were determined. These account for 203 of the approximately 300 amino acid residues of this protein. Several sites of carbohydrate attachment were observed.

scholarly journals Structural studies on bovine γ-crystallin

1971 ◽  
Vol 121 (3) ◽  
pp. 453-459 ◽  
Author(s):  
L. R. Croft ◽  
S. G. Waley
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Polypeptide Chain ◽  
Amino Acid Sequences ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Cysteine Residues ◽  
Lens Protein ◽  
Structural Studies ◽  
Protein Amino Acid

The amino acid sequences around the cysteine residues in the lens protein, γ-crystallin, were studied. Fraction II of the γ-crystallin from calf lens (Björk, 1964) was used. The protein was oxidized with performic acid and then hydrolysed with trypsin. Six peptides containing cysteic acid were isolated. One of the peptides contained three residues of cysteic acid and the others contained one residue of cysteic acid. We conclude that there are eight unique residues of cysteic acid in the oxidized protein. Amino acid analysis suggests that there are also eight residues of cysteic acid in the molecule, which thus contains only one polypeptide chain.

scholarly journals Preparation and properties of creatine kinase from the breast muscle of normal and dystrophic chicken (Gallus domesticus)

1970 ◽  
Vol 120 (1) ◽  
pp. 177-185 ◽  
Author(s):  
B. P. Roy ◽  
J. F. Laws ◽  
A. R. Thomson
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Breast Muscle ◽  
Chicken Breast ◽  
Kinetic Properties ◽  
Thiol Groups ◽  
Dystrophic Muscle ◽  
Peptide Maps ◽  
Dystrophic Chicken

1. The purification of creatine kinase from normal and genetically dystrophic chicken breast muscle is described. Enzyme recovery was significantly lower from dystrophic muscle. 2. Both enzymes had the same number of reactive and total thiol groups and had similar specific activities and similar amino acid compositions. 3. No significant differences were observed in sedimentation, electrophoretic or kinetic properties. 4. Peptide `maps' showed no significant differences, and electrophoresis of partial acid hydrolysates of the labelled enzymes suggested that corresponding amino acid sequences around all the thiol groups were very similar. 5. The enzymes showed identical temperature stabilities. 6. No significant differences between the enzymes from normal and dystrophic muscle were observed.

scholarly journals A novel giant secretion polypeptide in Chironomus salivary glands: implications for another Balbiani ring gene.

1985 ◽  
Vol 101 (3) ◽  
pp. 1044-1051 ◽  
Author(s):  
W Y Kao ◽  
S T Case
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Salivary Glands ◽  
Cyanogen Bromide ◽  
Tryptic Peptide ◽  
Amino Acid Sequences ◽  
The Other ◽  
Balbiani Ring ◽  
Sequence Organization ◽  
Peptide Maps

Chironomus salivary glands contain a family of high Mr (approximately 1,000 X 10(3)) secretion polypeptides thought to consist of three components: sp-Ia, sp-Ib, and sp-Ic. The use of a new extraction protocol revealed a novel high Mr component, sp-Id. Results of a survey of individual salivary glands indicated that sp-Id was widespread in more than a dozen strains of C. tentans and C. pallidivittatus. Sp-Id was phosphorylated at Ser residues, and a comparison of cyanogen bromide and tryptic peptide maps of 32P-labeled polypeptides suggested that sp-Ia, sp-Ib, and sp-Id are comprised of similar but nonidentical tandemly repeated amino acid sequences. We concluded that sp-Id is encoded by an mRNA whose size and nucleotide sequence organization are similar to Balbiani ring (BR) mRNAs that code for the other sp-I components. Furthermore, parallel repression of sp-Ib and sp-Id synthesis by galactose led us to hypothesize that both of their genes exist within Balbiani ring 2.

scholarly journals The binding and catalytic activities of forms of ligandin after modification of its thiol groups

1979 ◽  
Vol 177 (2) ◽  
pp. 433-439 ◽  
Author(s):  
T Carne ◽  
E Tipping ◽  
B Ketterer
Keyword(s):  
Thiol Group ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Thiol Groups ◽  
Glutathione S Transferase ◽  
Catalytic Activities ◽  
Nitrobenzoic Acid ◽  
Number Of Binding Sites ◽  
The Common ◽  
Hydrophobic Ligand

Ligandin (glutathione S-transferase B, EC 2.5.1.18)was treated with p-mercuribenzoate, N-(4-dimethylamino-3,5-dinitrophenyl)-maleimide, 5,5,-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, iodoacetamide or iodoacetate. Although performic acid oxidation revealed the presence of four cysteines, p-mercuribenzoate and N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, the most effective of the reagents studied, reacted with only three residues. N-Ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid) each reacted with two cysteines: iodoacetamide reacted with only one cysteine and iodoacetate was essentially unreactive. Modification of three thiol groups decreased both the enzymic and binding activities of ligandin although the number of binding sites was unaffected. Modification of only one or two of the thiol groups had little effect on the ligandin activities. It therefore appears that there is a thiol group in the common hydrophobic-ligand- and substrate-binding site of ligandin. Ligandin was separated into two fractions on CM-cellulose. Both fractions gave the same results with p-mercuribenzoate and iodoacetamide.

scholarly journals Determination of Sulfur Amino Acids in Foods as Related to Bioavailability

2008 ◽  
Vol 91 (4) ◽  
pp. 907-913 ◽  
Author(s):  
Shane M Rutherfurd ◽  
Paul J Moughan
Keyword(s):  
Methionine Sulfoxide ◽  
Performic Acid ◽  
Cysteic Acid ◽  
Acid Oxidation ◽  
Separate Determination ◽  
Sulfur Amino Acids ◽  
Methionine Sulfone ◽  
Quantitative Impact ◽  
Selection Of

Abstract During the processing of feedstuffs and foods, methionine can be oxidized to methionine sulfoxide and methionine sulfone, and cysteine can be oxidized to cysteic acid. Methionine sulfone and cysteic acid are nutritionally unavailable, but methionine sulfoxide can be utilized, at least to some degree. The degree of utilization depends on the levels of methionine, cysteine, and methionine sulfoxide in the diet, but there is no consensus in the literature on the quantitative impact of these dietary constituents on methionine sulfoxide utilization. Methionine and cysteine are most often determined after quantitative oxidation to methionine sulfone and cysteic acid, respectively, using performic acid oxidation prior to hydrolysis. However, this method may overestimate the methionine content of processed foods, as it will include any methionine sulfoxide and methionine sulfone present. A selection of analytical methods has been developed to allow the separate determination of the 3 oxidized forms of methionine, the merits of which are discussed in this review. An additional consideration for determining methionine and cysteine bioavailability is that not all dietary methionine and cysteine is digested and absorbed from the small intestine. Selected methods designed to determine the extent of digestion and absorption are discussed. Finally, a concept for a new assay for determining methionine bioavailability, which includes determining the digestibility of methionine and methionine sulfoxide as well as the utilization of methionine sulfoxide, is presented.

scholarly journals Disulphide bridges of the heavy chain of human immunoglobulin G2

1971 ◽  
Vol 121 (2) ◽  
pp. 217-225 ◽  
Author(s):  
C. Milstein ◽  
B. Frangione
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Heavy Chain ◽  
Amino Acid Sequences ◽  
Cysteic Acid ◽  
Partial Reduction ◽  
Variable Part ◽  
One Dimensional ◽  
Heavy Chains ◽  
Dimensional Electrophoresis

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the γ2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of γ2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the γ2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.

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