muramidase and sodium-borohydride

Topics: Borohydrides; Catalysis; Crystallization; Gold; Metal Nanoparticles; Muramidase; Nitrophenols; Oxidation-Reduction

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.

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

The amino acids in the peptidoglycan of Fusobacterium nucleatum Fev 1 are D-glutamic acid, meso-lanthionine, and D- (42%) and L-alanine (58%). About 70% of the lanthionine residues were not susceptible to dinitrophenylation, evidently because they are involved in cross-linkages. Consequently, lysozyme digestion of the peptidoglycan yielded 20 to 25% uncross-linked disaccharide tri- and tetrapeptides. A chemical analysis of isolated glycopeptides indicated that the structure of the building block of this peptidoglycan is N-acetylgucosamine-N-acetylmuramic acid-L-alanine-D-glutamic acid-meso-lanthionine(-D-alanine). I present evidence which supports the classification of the F. nucleatum Fev 1 peptidoglycan as a new A1 delta, directly cross-linked, meso-lanthionine-containing peptidoglycan.

Topics: Alanine; Bacteroidaceae; Borohydrides; Dinitrophenols; Glutamates; Glutamic Acid; Glycopeptides; Hydrazines; Muramidase; Peptidoglycan