Hospital Diet

The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein-N-acetylglucosamine and alpha-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn(2+) ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.

Section: Methodsmentioning

confidence: 99%

Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme

Kitchen¹,

Andrews²

1974

“…5,5'-Dithiobis-(2-nitrobenzic acid) (Nbs2) This was the BDH Chemicals Ltd. product recrystallized from acetic acid. The Nbs2resulting from cleavage of Nbs22was monitored spectrophotometrically by using Cary 15 and 16K spectrophotometers, thermostatically controlled at 25.0'C, and C412= 1.36 x 104 (Ellman, 1959) was used to calculate its concentration.…”

Section: A-n-benzoyl-l-arginine Ethyl Estermentioning

confidence: 99%

“…The first study of this type was made by Barrett & Seligman (1952), who used 2,2'-dihydroxy-6,6'-dinaphthyl disulphide to demonstrate proteinbound thiol groups histochemically. Ellman (1958) reported the use of bis-(p-nitrophenyl) disulphide for thiol-group determination and later (Ellman, 1959) enhanced its water solubility by introduction of carboxyl groups into the benzene rings. The extensive use of Ellman's (carboxylated) reagent, 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) in protein-modification studies has been reviewed (Glazer, 1970).…”

mentioning

confidence: 99%

The reaction of papain with Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoate) dianion]

Brocklehurst

Kierstan

Little

1972

1. The report by Robyt et al. (1971) that the 2-nitro-5-mercaptobenzoate dianion (Nbs(2-)) produced by reaction of papain with the 5,5'-dithiobis-(2-nitrobenzoate) dianion (Nbs(2) (2-); Ellman's reagent) cleaves the three disulphide bonds in papain is shown to be incorrect. 2. When partially active papain containing approx. 0.4 mol of thiol/mol of protein is incubated with excess of Nbs(2) (2-) at pH8, Nbs(2) (2-) reacts with the protein in an amount stoicheiometric with the cysteinyl thiol group of papain to produce Nbs(2-) in an amount stoicheiometric with the original papain cysteinyl thiol group, and the catalytically inactive mixed disulphide, papain-Nbs(-). 3. Papain catalyses the hydrolysis of Nbs(2) (2-) at pH10.5 probably by nucleophilic catalysis involving the enzyme's thiol group. 4. These results cast very serious doubts on the claim by Robyt et al. (1971) to have established a new general method for the determination of cystinyl disulphide residues in proteins.

“…Free thiol groups. Measurement by the method of Ellman (1959) gave approx. 0.3 SH groups/mol of al,-globulin.…”

Section: Analytical Resultsmentioning

confidence: 99%

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

Gordon¹,

Dykes²

1972

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.