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Instability of DTNB-treated globin or haemoglobin
Australian Journal of Biological Sciences
|July 1, 1976
Summary
Human globin reacts with DTNB, but isolation methods cause unwanted disulfide bond formation. This contrasts with other thiol-blocking reagents, highlighting DTNB
Area of Science:
- Biochemistry
- Protein Chemistry
- Molecular Biology
Background:
- Human hemoglobin contains reactive thiols at beta93 positions.
- 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reacts with these thiols.
- Standard protein isolation techniques can induce artifactual disulfide bonds.
Purpose of the Study:
- To investigate the reaction of human globin with DTNB.
- To characterize the stability of DTNB-modified globin during isolation.
- To compare DTNB modification with other thiol-blocking reagents.
Main Methods:
- Reaction of human globin with DTNB.
- Acetone-HCl precipitation for protein isolation.
- Urea-thiol buffer chromatography on carboxymethylcellulose.
- Amino acid analysis and peptide mapping.
- Gel filtration and SDS-PAGE for size determination.
Main Results:
- DTNB modifies human globin at beta93 thiols.
- Acetone-HCl treatment leads to disulfide interchange and oxidation of masked thiols.
- Globin treated with iodoacetic acid or N-ethylmaleimide remains stable.
- Derivatized globin chains and disulfide bond locations were identified.
Conclusions:
- DTNB modification of human globin is susceptible to artifactual disulfide bond formation during isolation.
- Reagents like iodoacetic acid and N-ethylmaleimide offer more stable thiol modification.
- Chromatographic and electrophoretic methods can characterize modified globin chains and disulfide bonds.
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