Menashi A. Cohenford

Nonenzymatically Galactosylated Serum Albumin in a Galactosemic Infant

Galactosemia is a molecular disease of genetic origin that is characterized by a deficiency of galactose-1-phosphate uridyltransferase.1 , 2 The resulting severe impairment of galactose metabolism ...

Nonenzymatic Glycosylation of Human Igg: In Vitro Preparation*

Incubation of human serum with either D- (1-14C) galactose (5 mM), D- (1-14C) glucose (5 mM) or L- (1-14C) fucose (5 mM) in vitro for 7 days under physiological conditions resulted in the accumulation of radioactivity into trichloroacetic acid precipitable material. Separation of the serum proteins by Sephadex G-200 chromatography, revealed the association of radioactivity with the albumin fraction (95%) and to a lesser extent with IgG (4%) and IgM (1%). D-galactose glycosylated purified human IgG at 2 to 3 fold the rate of D-glucose of L-fucose. The rate of glycose incorporation into IgG increased parabolically with increasing pH and temperature of incubation, and followed a first order dependence with either the glycose or the IgG concentration. The post-translational modification of IgG through nonenzymatic glycosylation may affect its immunological properties in clinical conditions associated with increased blood sugar concentrations.

Inhibitory effect of gold nanoparticles on the D-ribose glycation of bovine serum albumin

Formation of advanced glycation end products (AGEs) by nonenzymatic glycation of proteins is a major contributory factor to the pathophysiology of diabetic conditions including senile dementia and atherosclerosis. This study describes the inhibitory effect of gold nanoparticles (GNPs) on the D-ribose glycation of bovine serum albumin (BSA). A combination of analytical methods including ultraviolet–visible spectrometry, high performance liquid chromatography, circular dichroism, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were used to determine the extent of BSA glycation in the presence of citrate reduced spherical GNPs of various sizes and concentrations. GNPs of particle diameters ranging from 2 nm to 20 nm inhibited BSA’s AGE formation. The extent of inhibition correlated with the total surface area of the nanoparticles. GNPs of highest total surface area yielded the most inhibition whereas those with the lowest total surface area inhibited the formation of AGEs the least. Additionally, when GNPs’ total surface areas were set the same, their antiglycation activities were similar. This inhibitory effect of GNPs on BSA’s glycation by D-ribose suggests that colloidal particles may have a therapeutic application for the treatment of diabetes and conditions that promote hyperglycemia.

A fluorometric method for quantitating the enzymatic hydrolysis of fucose from porcine submaxillary mucin (A

Abstract A fluorometric method for quantitating the amount of α- l -fucose enzymatically released from porcine submaxillary mucin (A+) has been developed. The released fucose is oxidized with fucose dehydrogenase and NAD, and the NADH produced, in conjunction with diaphorase is then used to convert the nonfluorescent dye, resazurin, to the highly fluorescent product, resorufin. The method can detect as little as 0.05 nmol of α- l -fucose. α- l -Fucosidase from the mollusc Mercenaria mercenaria is employed for the hydrolysis reaction. The enzymatic release of fucose is maximal to pH 2.85 and, the reaction rate is linearly proportional to incubation time for 20 min under the conditions employed. The presence of fucose in the reaction mixture, after hydrolysis has occurred, is demonstrated by paper chromatography. This procedure provides a rapid, simple, and specific method for quantitating the release of α- l -fucose from porcine submaxillary mucin type A+.

Purification and properties of two forms of α-l-fucosidase from Turbo cornutus

Abstract 1. 1. Two forms of α- l -fucosidase were isolated from the gastropod, Turbo cornutus and their properties studied. 2. 2. Fucosidase I (pI 6.78) and Fucosidase II (pI 5.5) were purified by a scheme which included Sephadex G-200 column chromatography, DEAE-Sephadex column chromatography, heat treatment, CM-cellulose column chromatography and preparative isoelectric focusing. 3. 3. The purified forms were substantially free of all other glycosidase contaminations and had specific activity approximately 73 and 65 fold of the starting material. 4. 4. The pH activity profile of Fucosidase I displayed a pH optimum at 4.0, and that of Fucosidase II showed a broad optimum centered at pH 3.6. 5. 5. Fucosidase I was thermolabile and lost greater than 90% of its activity when incubated at 55°C for 15 min. Fucosidase II was thermostable, and lost less than 30% of its initial activity under identical incubation conditions. 6. 6. Both enzymes had an apparent molecular weight of 230,000 ± 20,000 and were inhibited by Hg2+, l -fucose and l -galactose. 7. 7. The Km values of Fucosidase I and II for the substrate p- nitrophenyl-α- l -fucopyranoside were 0.38 and 0.14 mM, respectively.

Nonenzymatic Galactosylation of Proteins and Galactosemia

Hereditary galactosemia due to galactose-l-phosphate uridyl transferase deficiency produces severe illness often leading to death in the neonate- Surviving infants, if dietary galactose is not restricted, develop cataracts, jaundice and irreversible mental retardation. Presently, in patients with galactosemia, the relationship between elevated tissue concentrations of galactose and pathological complications has remained obscure. We suggested that galactosemia pathophysiology may be related to the nonenzymatic galactosylation of proteins in vivo.