TY - JOUR

T1 - Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4'-dithiodipyridine.

AU - Hansen, Rosa E

AU - Østergaard, Henrik

AU - Nørgaard, Per

AU - Winther, Jakob R

N1 - Keywords: Animals; Borohydrides; Carboxypeptidases; Cattle; Chromatography, High Pressure Liquid; Cysteine; Disulfides; Dithiothreitol; Muramidase; Oxidation-Reduction; Proteins; Pyridines; Ribonuclease, Pancreatic; Saccharomyces cerevisiae Proteins; Serum Albumin; Sulfhydryl Compounds; Toluene

PY - 2007

Y1 - 2007

N2 - Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure. Udgivelsesdato: 2007-Apr-1

AB - Experimental determination of the number of thiols in a protein requires methodology that combines high sensitivity and reproducibility with low intrinsic thiol oxidation disposition. In detection of disulfide bonds, it is also necessary to efficiently reduce disulfides and to quantify the liberated thiols. Ellman's reagent (5,5'-dithiobis-[2-nitrobenzoic acid], DTNB) is the most widely used reagent for quantification of protein thiols, whereas dithiothreitol (DTT) is commonly used for disulfide reduction. DTNB suffers from a relatively low sensitivity, whereas DTT reduction is inconvenient because the reagent must be removed before thiol quantification. Furthermore, both reagents require a reaction pH > 7.0 where oxidation by ambient molecular oxygen is significant. Here we describe a quick and highly sensitive assay for protein thiol and dithiol quantification using the reducing agent sodium borohydride and the thiol reagent 4,4'-dithiodipyridine (4-DPS). Because borohydride is efficiently destroyed by the addition of acid, the complete reduction and quantification can be performed conveniently in one tube without desalting steps. Furthermore, the use of reverse-phase high-performance liquid chromatography for the thiol quantification by 4-DPS reduces the detection limit to the picomolar range (equivalent to 1 microg of a 50-kDa protein containing 1 thiol) while at the same time maintaining low pH throughout the procedure. Udgivelsesdato: 2007-Apr-1

U2 - 10.1016/j.ab.2007.01.002

DO - 10.1016/j.ab.2007.01.002

M3 - Journal article

C2 - 17286954

VL - 363

SP - 77

EP - 82

JO - Analytical Biochemistry

JF - Analytical Biochemistry

SN - 0003-2697

IS - 1

ER -