Determination and derivatization of protein thiols by n-octyldithionitrobenzoic acid.
n-Octyl-5-dithio-2-nitrobenzoic acid (ODNB), the reaction product of n-octane-1-thiol and Ellman's reagent, can be isolated in crystalline form. Like Ellman's reagent, ODNB can be used for the titration of SH groups in proteins, but with considerable advantages: Due to the absence of one negative charge and the presence of a lipophilic hydrocarbon chain, titrations of protein thiols are significantly faster. Normally, endpoints are reached after 5-30 min, while the corresponding reaction with Ellman's reagent may take 1-2 h. Another advantage of ODNB is that it finds access to thiol groups which normally remain undetected. With myosin S1, for example, ODNB reacts with a thiol group which is not reactive with Ellman's reagent. Although ODNB has detergent properties, reaction of this thiol group as a consequence of denaturation seems unlikely, because ODNB was used below its critical micelle concentration and all other thiols of myosin S1 remained buried. The n-octylthiol derivatives of proteins formed by reaction with ODNB can be used for transient protection of thiols, for example, during purification procedures, or for a reversible blocking of SH groups involved in biochemical reactions.
Faulstich
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Max-Planck-Institut für Medizinische Forschung, Heidelberg, Germany.
Tews
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Heintz
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D000818:Animals; D004220:Disulfides; D004228:Dithionitrobenzoic Acid; D005069:Evaluation Studies as Topic; D015879:Myosin Subfragments / Q000032:analysis / Q000737:chemistry; D009579:Nitrobenzoates; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011817:Rabbits; D013438:Sulfhydryl Compounds / Q000032:analysis* / Q000737:chemistry; D013439:Sulfhydryl Reagents
D004220:Disulfides; D015879:Myosin Subfragments; D009579:Nitrobenzoates; D011506:Proteins; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C080134:n-octyl-5-dithio-2-nitrobenzoic acid; D004228:Dithionitrobenzoic Acid