scholarly journals Instability of DTNB-Treated Globin or Haemoglobin

1976 ◽  
Vol 29 (3) ◽  
pp. 181
Author(s):  
NG Baptist ◽  
AR Nash ◽  
EOP Thompson
Keyword(s):  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Iodoacetic Acid ◽  
Thiol Groups ◽  
Native Form ◽  
Human Haemoglobin ◽  
Globin Chains ◽  
Disulphide Bonds ◽  
Almost All

Human haemoglobin or globin in its native form reacts with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with uptake of two 3-carboxylato-4-nitrothiophenol groups, one for each of the reactive thiols at the {393 positions. Attempts to isolate the DTNB-treated globin by the acetone-Hel method, which unfolds the protein chains, result in disulphide interchange and oxidation of almost all the uncoupled 'masked' thiol groups. This modification is in marked contrast to the stability of haemoglobin or globin treated with reagents such as iodoacetic acid or N-ethylmaleimide that do not form disulphide bonds in blocking the thiol groups. The derivatized globin chains have been separated by urea-thiol buffer chromatography on carboxymethylcellulose columns. Amino acid analysis and peptide mapping established the presence and location of disulphide bonds, whilst gel filtration in urea buffers and sodium dodecyl sulphate acrylamide gel electrophoresis defined the size of the products.

scholarly journals Tamm–Horsfall urinary glycoprotein. The subunit structure

1970 ◽  
Vol 120 (2) ◽  
pp. 425-432 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Subunit Molecular Weight ◽  
Disulphide Bonds ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. Subunit molecular weights of 76000–82000 were obtained for native and alkylated Tamm–Horsfall glycoprotein by gel filtration on Sephadex G-200 in the presence of sodium dodecyl sulphate. 2. A further estimate of the subunit molecular weight of 79000±4000 was obtained by disc gel electrophoresis in sodium dodecyl sulphate. 3. A minimum value of the chemical molecular weight of 79000±6000 was obtained from the number of N-terminal amino acids released by cyanogen bromide cleavage of the glycoprotein. 4. Similar values were obtained for the subunit molecular weight of Tamm–Horsfall glycoprotein from patients with cystic fibrosis. 5. On ultracentrifugation both in 1.0% sodium dodecyl sulphate and in 70% formic acid, Tamm–Horsfall glycoprotein sedimented as a single component, slightly faster than serum albumin. 6. On reduction of the disulphide bonds the same subunit molecular weight was obtained, which suggested that these bonds are intrachain.

scholarly journals Purification of two hexosaminidases from human kidney

1977 ◽  
Vol 163 (1) ◽  
pp. 133-140 ◽  
Author(s):  
D V Marinkovic ◽  
J N Marinkovic
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Human Kidney ◽  
Protein Band ◽  
Iodoacetic Acid ◽  
Homogeneous State ◽  
Molecular Weights ◽  
Hexosaminidase A

Hexosaminidase forms A and B were isolated from human kidney in a homogeneous state as demonstrated by electrophoretic and enzymic criteria. The enzymes were stable for at least 18 months when stored at -20 degrees C in 0.025 M-phosphate buffer, pH 6.5. The molecular weights of forms A and B were estimated by gel filtration to be 111 000 +/- 1500 and 114 000 +/- 1600 respectively. The molecular weights of hexosamidase A and B subunits were determined by using polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. Hexosaminidase A dissociated into one subunit with mol.wt. 68 000. Hexosaminidase B dissociated into three subunits with mol. wts. 100 000, 68 000 and 37000 respectively, and one protein band of mol.wt. 140 000. After treatment of hexosaminidases A and B with iodoacetic acid, the molecular weights of the carboxymethylated polypeptide subunits were also estimated. Carboxymethylated hexosaminidase A dissociated into one major subunit of mol.wt. 18 000 and two other protein bands of mol.wts. 65 000 and 100 000. Carboxymethylated hexosaminidase B dissociated into one major subunit for mol.wt. 19 000 and an additional band of mol.wt. 37 000. The Km of the enzymes for the synthetic substrate p-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside was 0.8 mM. Both enzymes were inhibited or activated by various metal ions. Double pH optima for the enzymes were found at pH 4.5 and 4.8.

Haemodialysis-associated amyloidosis: β2-microglobulin alone or associated with globin chains?

1987 ◽  
Vol 73 (5) ◽  
pp. 515-518 ◽  
Author(s):  
A. Argiles ◽  
G. Mourad ◽  
Claude Axelrud-Cavadore ◽  
J. Derancourt ◽  
J. Jauregui Adell ◽  
...  
Keyword(s):  
High Performance ◽  
Amyloid Fibrils ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Β2 Microglobulin ◽  
Maintenance Haemodialysis ◽  
Globin Chains ◽  
Amyloid Deposits ◽  
Mechanisms Of Formation

1. The protein constituents of amyloid fibrils were characterized in amyloid deposits extracted from surgical material obtained from a 66-year-old patient undergoing maintenance haemodialysis and operated for a carpal tunnel syndrome. 2. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis disclosed the presence of bands at 12 and 14 kDa. Two-dimensional electrophoresis and Western blotting confirmed that the proteins were β2-microglobulin (β2M) and globin chains. 3. When the effluent of high-performance gel filtration chromatography corresponding to molecular masses of 10–15 kDa was subjected to Edman degradation, only one amino acid residue was found at each step. The 18 residues determined corresponded to the N-terminal sequence of β2M. 4. Although globin chains were clearly present in the amyloid material, they were not accessible for sequence determination. The identification of the other protein constituents present in the amyloid material, along with β2M, should provide a better understanding of haemodialysis-associated amyloidosis, the mechanisms of formation of which have not yet been completely determined.

scholarly journals Studies on the Structure and Subunit Composition of Human Antihaemophilic Factor

1977 ◽  
Author(s):  
J. J. Gorman
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Protein S ◽  
Molecular Forms ◽  
Major Band ◽  
Human Factor Viii ◽  
Von Willebrand

Human antihaemophilic factor has been purified by hydroxylapatite chromatography following precipitation from plasma and gel filtration on Sepharose 6B.Application to hydroxyiapatite was in 0.02 M tris HCl (pH 7.35) – 0.14 M NaCl and after washing with 5mM phosphate (pH 6.8) – 0.1 M NaCI the antihaemophilic factor was eluted with 0. 1M phosphate (pH 6.8) – 0.1M NaCl. Factor VIII coagulant activity, factor VIII related antigen and von Willebrand factor activity eluted simultaneously.The protein(s) had a molecular weight in excess of 500,000 and multiple subunits as shown by electrophoresis in 5% acrylamide gels containing sodium dodecyl sulphate;without reduction the protein failed to enter these gels but following reduction multiple bands were observed, the major band had a molecular weight around 200,000.Thin layer peptide mapping demonstrated structural inter-relationship between the 200,000 dalton protein and three of the smaller species, however, two other unrelated smaller species were evident.It is apparent from these findings that human factor VIII may exist as multiple molecular forms due to heterogeneity of one subunit (MW around 200,000) and the molecular structure may include other smaller non-identical subunits. The structure-function relationships of these subunits remains to be elucidated.

Identification of high-affinity calmodulin-binding proteins in rat liver

1987 ◽  
Vol 252 (3) ◽  
pp. C277-C284 ◽  
Author(s):  
R. M. Hanley ◽  
J. R. Dedman ◽  
S. Shenolikar
Keyword(s):  
Rat Liver ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Apparent Molecular Weight ◽  
Convincing Evidence ◽  
Calmodulin Binding ◽  
High Affinity

The Ca2+-dependent binding of [125I]calmodulin (CaM) to hepatic proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized to identify CaM binding or “acceptor” proteins or CAPs. Two proteins of apparent molecular weight of 60,000 (CAP-60) and 45,000 (CAP-45) comprised greater than 80% of the Ca2+-dependent CaM binding in rat liver cytosol. CAP-60 and CAP-45 were partially purified by a variety of chromatographic steps, including affinity chromatography on CaM Sepharose. CAP-60 possessed a native molecular size of 400,000, indicating it to be the CaM-binding “subunit” of a larger oligomeric complex. In contrast, CAP-45 was monomeric as judged by gel filtration. Neither CAP-60 nor CAP-45 possessed chromatographic properties consistent with known CaM-dependent enzymes reported in the literature. Two-dimensional peptide mapping provided convincing evidence that CAP-60 and CAP-45 were unrelated to other well-characterized CAPs, namely Ca2+ (CaM)-dependent protein kinase II, calcineurin, or the CaM-dependent cyclic nucleotide phosphodiesterase. The relative abundance and high affinity for CaM could suggest that these novel target proteins, CAP-60 and CAP-45, represent a dominant pathway for CaM action in the mammalian liver.

A preliminary study by gel filtration and ultracentrifugation of the interaction of bovine milk caseins with detergents

1968 ◽  
Vol 35 (3) ◽  
pp. 439-446 ◽  
Author(s):  
G. C. Cheeseman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Gel Filtration ◽  
Sedimentation Velocity ◽  
Disulphide Bonds ◽  
Reducing Conditions ◽  
Preliminary Study ◽  
The Individual

SummaryThe detergents sodium dodecyl sulphate and octyl phenoxy polyethoxyethanol interact with casein and cause dissociation of the high-molecular-weight casein aggregates. It is presumed that the detergent binds with hydrophobic regions in the casein molecule. The size of the complexes formed between detergents and αs1-casein, β-casein and κ-casein, as estimated by gel filtration and sedimentation velocity experiments, suggests that the caseins were complexed as monomers.During gel filtration under non-reducing conditions, detergent-κ-casein complexes were separated from other major components because of their conversion through formation of disulphide bonds into high-molecular-weight aggregates. This reaction, which did not occur in sedimentation velocity experiments, was presumably facilitated by the changes in the equilibrium between the individual caseins during gel filtration.Sedimentation velocity experiments showed that a ratio of about 40 detergent molecules to 1 casein molecule was required to give the smallest casein-detergent complex.

scholarly journals cis, cis-Muconate cyclase from Trichosporon cutaneum

1980 ◽  
Vol 191 (1) ◽  
pp. 37-43 ◽  
Author(s):  
A Gaal ◽  
H Y Neujahr
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Trichosporon Cutaneum ◽  
Thiol Groups ◽  
Divalent Metal Ions ◽  
Ph Optimum

The inducible enzyme catalysing the conversion of cis, cis-muconate to (+)-muconolactone was purified 300-fold from the yeast Trichosporon cutaneum, grown on phenol. The enzyme has a sharp pH optimum at pH 6.6. It reacts also with several monohalogen derivatives and with one monomethyl derivative of cis, cis-muconate, but not with cis, trans- or trans, trans-muconate or 3-carboxy-cis, cis-muconate. In contrast with the corresponding enzymes in bacteria, the yeast enzyme does not require added divalent metal ions for activity and is not inhibited by EDTA. The purified enzyme can be resolved into two peaks by isoelectric focusing. The two forms have pI 4.58 (cis, cis-muconate cyclase I) and pI 4.74 (cis, cis-muconate cyclase II), respectively. Each of these is homogenous on polyacrylamide-gel electrophoresis in the absence or presence of sodium dodecyl sulphate. The two enzyme forms have the same molecular weight (50000) as determined by gel filtration and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. They have the same Km value (25 microM) for cis, cis-muconate. They differ with respect to their content of free thiol groups. cis, cis-Muconate cyclase I contains one thiol group, essential for activity, but relatively stable upon storage. cis, cis-Muconate cyclase II contains two thiol groups that are readily oxidized during storage with concomitant loss of activity.

scholarly journals Mouse intracellular immunoglobulin M. Structure and identification of a free thiol group

1978 ◽  
Vol 175 (3) ◽  
pp. 959-967 ◽  
Author(s):  
Neil E. Richardson ◽  
Arnold Feinstein
Keyword(s):  
Plasma Cells ◽  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Thiol Group ◽  
Immunoglobulin M ◽  
Thiol Groups ◽  
Heavy Chains ◽  
Free Thiol ◽  
Free Thiol Group

Monomeric intracellular mouse immunoglobulin M (hereafter designated IgMs) was purified in milligram quantities from the plasma cells of mouse plasmacytoma MOPC 104E after lysis either in the presence or in the absence of iodoacetate. Peptide ‘mapping’ analysis of the IgMs after partial reduction and carboxy[14C]methylation to label the interchain disulphide bridges showed that the heavy–light bridge and the interheavy bridge present in the Cμ2 region were already formed at lysis. The cysteine residues in the C-terminal region of the heavy chains, which in pentameric IgM form an intersubunit bridge, had free thiol groups at lysis that were reversibly oxidized during isolation in the absence of iodoacetate, probably forming an intrasubunit inter-heavy-chain disulphide bridge. Isoelectric-focusing studies complemented the above findings, showing that all the intracellular IgMs carried free thiol groups that could be carboxymethylated at lysis, and that in non-alkylated preparations these had reversibly oxidized. On the basis of sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis intracellular μ-chains had a consistently lower apparent molecular weight than did secreted μ-chains, and the estimated difference could be accounted for by the known difference in carbohydrate content. We present evidence that in a position homologous to that of a complex oligosaccharide in the Cμ2 region of secreted human μ-chains there is a simple oligosaccharide in intracellular mouse μ-chains that becomes complex on secretion. On the basis of the above findings, we present a model for the mouse intracellular IgM subunit and suggest a mechanism for its assembly into secreted IgM pentamers.

scholarly journals Carboxymethylation of Thiol Groups in Ovalbumin: Implications for Proteins that Contain Both Thiol and Disulfide Groups

1982 ◽  
Vol 35 (2) ◽  
pp. 125 ◽  
Author(s):  
DM Webster ◽  
EOP Thompson
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Iodoacetic Acid ◽  
Cysteine Residues ◽  
Alkaline Ph ◽  
Thiol Groups ◽  
Nitrobenzoic Acid ◽  
Dodecyl Sulfate ◽  
Fold Excess

The cysteine residues of hen ovalbumin were S-carboxymethylated with non-radioactive iodoacetic acid under various conditions by altering the pH at which the protein was denatured in 8 M urea, by using different molar ratios of non-radioactive iodoacetic acid to cysteine and by varying the time at which carboxymethylation was commenced after denaturing conditions had been applied. Under the various conditions, the thiol groups were carboxymethylated to different extents, the residual thiol groups being measured by reaction with 5,5'-dithiobis(2-nitrobenzoic acid) in the presence of sodium dodecyl sulfate. When ovalbumin is carboxymethylated in alkaline urea, it unfolds slowly and the carboxymethylation is incomplete even with 150-fold excess iodoacetic acid. The known rapid thiol-disulfide exchange that occurs at alkaline pH values makes this method of carboxymethylation unsuitable as a preliminary step for blocking the native cysteine residues of ovalbumin before reduction and labelling the thiol groups formed by reduction of the disulfide bonds. Titration of the thiol groups of ovalbumin in 6 M guanidine hydrochloride or 1 % (w/v) sodium dodecyl sulfate at pH 8�2 with 5,5' -dithiobis(2-nitrobenzoic acid) is more rapid than in 8 M urea and these solvents would be preferable for studies of the disulfide-bonded sequences. Denaturation of ovalbumin in acidic 8 M urea is a very rapid process, and under mild acid conditions thiol-disulfide interchange is much slower. Subsequent carboxymethylation of the cysteine residues at alkaline pH with 150-fold excess iodoacetic acid results in complete carboxymethylation and the carboxymethylated ovalbumin can be reduced and labelled with radioactive iodoacetic acid with specific labelling of the half-cystine residues involved in the disulfide bond. The results are discussed in relation to the allocation of half-cystine residues in other protein systems that contain both thiol and disulfide groups.

scholarly journals Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis

1976 ◽  
Vol 153 (1) ◽  
pp. 79-87 ◽  
Author(s):  
N Maeda ◽  
N Tamiya
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Amino Acid Sequences ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Toxin B ◽  
Almost All

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 μg/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.

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