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.

Fractionation of nucleolar proteins by two-dimensional gel electrophoresis

1976 ◽  
Vol 54 (1) ◽  
pp. 9-14 ◽  
Author(s):  
G. Jackowski ◽  
D. Suria ◽  
C. C. Liew
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Nucleolar Proteins ◽  
Dodecyl Sulfate ◽  
Ph Range

Isolation of nucleolar proteins was obtained by dissociation in the presence of urea – guanidine hydrochloride, followed by high-speed centrifugation to remove nucleic acids. At least 31 fractions of nucleolar proteins were detected by isoelectrofocusing gel electrophoresis in the pH range 3.5–10. Following two-dimensional gel electrophoresis on sodium dodecyl sulfate – polyacrylamide slab gels, more than 100 components of nucleolar proteins were identified. Two-thirds of nucleolar proteins were located in the pH range 5–8 following isoelectrofocusing. The molecular weights of these classes of proteins were shown to be mostly 30 000 – 70 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis.

scholarly journals Characterization of erythropoietin produced by IW32 murine erythroleukemia cells

Blood ◽  
1984 ◽  
Vol 64 (2) ◽  
pp. 341-347 ◽  
Author(s):  
J Choppin ◽  
C Lacombe ◽  
N Casadevall ◽  
O Muller ◽  
P Tambourin ◽  
...  
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Cell Line ◽  
Mononuclear Cells ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Calf Serum ◽  
Sodium Metaperiodate ◽  
Dodecyl Sulfate ◽  
Murine Erythroleukemia ◽  
Cells Cultured

Abstract IW32 is a recently described murine erythroleukemia cell line that produces an erythropoietic factor similar to erythropoietin by in vivo and in vitro bioassays and without species specificity. Biochemical characteristics of IW32 erythropoietic factor and sheep or mouse plasma erythropoietins were compared. Murine colonies derived from erythroid colony-forming units (CFU-E) in plasma clot culture were used as the bioassay system. Both IW32 erythropoietic factor and sheep plasma erythropoietin were stable in the pH range of 3 to 10, after exposure to denaturing agents (8 mol/L urea, 4 mol/L guanidine hydrochloride, 1% sodium dodecyl sulfate), to a reducing agent (0.1 mol/L 2- mercaptoethanol) and to an oxidizing agent (5 mmol/L sodium metaperiodate). Only the combination of 0.1 mol/L 2-mercaptoethanol and 1% sodium dodecyl sulfate resulted in a significant loss of activity. IW32 erythropoietic factor and murine plasma erythropoietin were similarly precipitated by ethanol and ammonium sulfate. IW32 erythropoietic factor eluted as a single major peak after gel exclusion chromatography, with an estimated molecular weight of 45,000 daltons. Results were identical using supernatants from cultures in the presence of and absence of fetal calf serum. The supernatant of IW32 cells cultured without serum induced erythroid colonies after seven days on normal human bone marrow nonadherent mononuclear cells cultured in serum-free conditions. All these results made it very likely that IW32 cells produce an authentic erythropoietin. This cell line would be very useful for the study of murine erythropoietin.

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