scholarly journals Novel heparin degradation products. Isolation and characterization of novel disaccharides and oligosaccharides produced from heparin by bacterial degradation

1968 ◽  
Vol 108 (4) ◽  
pp. 647-654 ◽  
Author(s):  
Carl P. Dietrich
Keyword(s):  
Paper Chromatography ◽  
Uronic Acid ◽  
Degradation Products ◽  
Gel Filtration ◽  
Present Knowledge ◽  
Bacterial Degradation ◽  
Isolation And Characterization ◽  
Flavobacterium Heparinum

1. Heparin was degraded by enzymes of adapted Flavobacterium heparinum. Several degradation products were separated by combined Sephadex-gel filtration and paper chromatography, and chemically analysed. 2. These products were identified as glucosamine 2,6-disulphate, saturated disaccharides constituted of uronic acid and glucosamine and containing two and three sulphate residues, and tetra- and hexa-saccharides with the same basic disaccharide units. 3. The implications of these findings with respect to the present knowledge of heparin structure and its enzymic degradation are discussed.

A uronic acid isomerase in Flavobacterium heparinum

1970 ◽  
Vol 48 (2) ◽  
pp. 164-169 ◽  
Author(s):  
James C. Karapally ◽  
Carl P. Dietrich
Keyword(s):  
Hyaluronic Acid ◽  
Equilibrium Point ◽  
Galacturonic Acid ◽  
Uronic Acid ◽  
Glucuronic Acid ◽  
Chondroitin Sulfates ◽  
Isolation And Characterization ◽  
Flavobacterium Heparinum

The isolation and characterization of a uronic acid isomerase from F. heparinum is described. The enzyme converts glucuronic acid and galacturonic acid to fructuronic acid and tagaturonic acid. The equilibrium point of the reaction is affected by buffers. In the presence of borate, 75% of glucuronic acid is converted to fructuronic acid. In the presence of phosphate, the equilibrium is reached when 31% of glucuronic acid is converted to fructuronic acid. Conversely, fructuronic acid and tagaturonic acid are converted to glucuronic acid and galacturonic acid, respectively. Monosaccharides derived from heparin and chondroitin sulfates do not affect the activity of the isomerase, in contrast to the monosaccharides from hyaluronic acid which have marked inhibitory (or diluting) activity upon the enzyme. The role of this enzyme in the metabolism of mucopolysaccharides is discussed.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

scholarly journals α-Crystallin. The isolation and characterization of distinct macromolecular fractions

1971 ◽  
Vol 124 (2) ◽  
pp. 337-343 ◽  
Author(s):  
Abraham Spector ◽  
Lu-Ku Li ◽  
Robert C. Augusteyn ◽  
Arthur Schneider ◽  
Thomas Freund
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Chemical Difference ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Different Populations ◽  
Molecular Weight Fractions

α-Crystallin was isolated from calf lens periphery by chromatography on DEAE-cellulose and gel filtration. Three distinct populations of macromolecules have been isolated with molecular weights in the ranges approx. 6×105−9×105, 0.9×106−4×106and greater than 10×106. The concentration of macromolecules at the molecular-weight limits of a population are very low. The members of the different populations do not appear to be in equilibrium with each other. Further, in those molecular-weight fractions investigated, no equilibrium between members of the same population was observed. The population of lowest molecular weight comprises 65–75% of the total material. The amino acid and subunit composition of the different-sized fractions appear very similar, if not identical. The only chemical difference observed between the fractions is the presence of significant amounts of sugar in the higher-molecular-weight fractions. Subunit molecular weights of approx. 19.5×103and 22.5×103were observed for all α-crystallin fractions.

IMPURITY PROFILING IMPURITY PROFILING OF THIAMINE HYDROCHLORIDE INJECTION BY RP-HPLC AND CHARACTERIZATION OF DEGRADATION PRODUCT BY LC-MS/MS/QTOF

2020 ◽  
pp. 151-161
Author(s):  
SRINIVASU KONDRA ◽  
BAPUJI A. T. ◽  
D. GOWRI SANKAR ◽  
POTTURI MURALI KRISHNAM RAJU
Keyword(s):  
Degradation Product ◽  
Degradation Products ◽  
Hplc Method ◽  
Thiamine Hydrochloride ◽  
Injectable Formulation ◽  
Hyphenated Technique ◽  
Isolation And Characterization ◽  
Impurity Profiling ◽  
Rp Hplc

Objective: To propose a comprehensive, simple, and affordable RP-HPLC method for impurity profiling and characterization of unknown degradation products of thiamine hydrochloride injectable formulation. Methods: The chromatographic separation employs gradient mode using the octadecyl silane column using a mobile phase consisting of phosphate buffer with ion pair reagent, acetonitrile, and methanol delivered flow rate at 1.2 ml/min. The detection was carried out at 248 nm using empower software. LC-MS/MS/QTOF hyphenated technique was used for isolation and characterization of unknown degradation impurity. The performance of the method was systematically validated as per ICH Q2 (R1) guidelines. Results: Degradation product observed in accelerated stability was characterized by LC-MS/MS/QTOF hyphenated technique and found m/z value 351.1604 and postulated as an oxidative degradation product of thiamine due to excipient interaction. The validated method was sensitive, selective, and specific data proves the method is precise and accurate from LOQ to 150% level and results are within 95-108% and less than 4.5% RSD. The developed method is linear from 0.03-58.83 µg/ml with a correlation coefficient of more than 0.990 and LOD and LOQ value ranged from 0.03 to1.51 μg/ml. Conclusion: An efficient RP-HPLC method for impurity profiling of thiamine injectable formulation was successfully developed and unknown degradation product observed instability condition samples characterized by LC-MS/MS/QTOF technique. The validated method can be successfully employed for the impurity profiling of thiamine injectable in the quality control department.

scholarly journals Isolation and characterization of a novel haemoprotein b-559 from Bengal gram (Cicer arietinum)

1987 ◽  
Vol 243 (3) ◽  
pp. 723-728 ◽  
Author(s):  
C S Ramadoss ◽  
B C Shenoy ◽  
A Borthakur
Keyword(s):  
Gel Filtration ◽  
Molecular Size ◽  
Soret Band ◽  
Beta Band ◽  
Photosynthetic Membrane ◽  
Isolation And Characterization ◽  
Bengal Gram ◽  
Apparent Homogeneity ◽  
Reduced State

A haemoprotein was purified to apparent homogeneity from Bengal-gram seeds. The purified protein exhibited an absorption maximum at 412 nm (Soret band) that upon reduction with dithionite gave rise to a shift in the Soret band to 426 nm with concomitant appearance of an alpha-band at 559 nm and a beta-band at 530 nm. In the reduced state the Bengal-gram haemoprotein showed reactivity towards CO, nitrite and hydroxylamine. SDS/polyacrylamide-slab-gel electrophoresis showed that the haemoprotein has Mr 78,000. Gel-filtration and ultracentrifugal analyses suggest that the Bengal-gram haemoprotein is oligomeric in nature. Since it differs from photosynthetic membrane cytochrome b-559 in solubility in buffer, in reactivity towards CO and in molecular size, it appears to be a novel haemoprotein b-559.

scholarly journals Isolation and characterization of glial filaments from human brain.

1978 ◽  
Vol 78 (2) ◽  
pp. 426-440 ◽  
Author(s):  
J E Goldman ◽  
H H Schaumburg ◽  
W T Norton
Keyword(s):  
White Matter ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Neurofilament Proteins ◽  
Human Central Nervous System ◽  
Neurofilament Protein ◽  
Isolation And Characterization ◽  
Single Protein ◽  
Filament Protein

Intermediate (8--9 nm) filaments of human central nervous system astrocytes were isolated from the gliosed white matter of cases of adrenoleukodystrophy (ALD). This hereditary lipidosis is characterized pathologically by demyelination, loss of axons, and replacement of the white matter of the caudal cerebrum by a glial scar. Glial filaments were composed largely of a single protein component with a mol wt of about 49,000 daltons. Smaller components (44,000--39,000 daltons) were detected in some samples, and appear to represent degradation products of the filament protein. Human neurofilaments were isolated from the normal frontal white matter of ALD cases by the standard myelin-free axon technique. Isolated glial and neurofilament proteins comigrated during acrylamide gel electrophoresis in SDS. Polypeptides resulting from cyanogen bromide cleavage of the two filament proteins were the same. Both proteins reacted with rabbit antisera raised against isolated bovine neurofilament protein and human glial fibrillary acidic protein.

scholarly journals Isolation and characterization of membrane proteins responsible for attachment of polyribosomes to rough microsomal fraction of rat liver

1977 ◽  
Vol 164 (1) ◽  
pp. 53-66 ◽  
Author(s):  
S Fujita ◽  
F Ogata ◽  
J Nakamura ◽  
S Omata ◽  
H Sugano
Keyword(s):  
Rat Liver ◽  
Protein Fraction ◽  
Microsomal Fraction ◽  
Binding Capacity ◽  
Gel Filtration ◽  
High Affinity ◽  
Isolation And Characterization ◽  
Microsomal Membranes ◽  
Equilibrium Centrifugation

A protein fraction which has a high affinity for polyribosomes was isolated from rough microsomal membranes of rat liver. The mode of polyribosome binding to this fraction (R-fraction) was studied by using CsCl equilibrium centrifugation and compared with that for stripped rough microsomal membranes. The following were found. (1) The polyribosome-binding cpacity of the R-fraction was heat-labile and sensitive to trypsin, and was suppressed by increasing KCl concentration and addition of 0.1 mM-aurintricarboxylic acid. (2) Of the four subfractions obtained by gel filtration of the R-fraction on a Sephadex G-200, only the R1-fraction, eluted at the void volume, showed a high affinity for polyribosomes. The polyribosome-binding capacity of the R1-fraction decreased with time on storage at 4 degrees C. (3) The R1-fraction contained three major proteins with mol. wts. 108,000, 99,000 and 65,000.

scholarly journals Isolation and characterization of lung connective-tissue glycoproteins

1982 ◽  
Vol 203 (3) ◽  
pp. 593-601 ◽  
Author(s):  
C Lafuma ◽  
M Moczar ◽  
L Robert
Keyword(s):  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lung Parenchyma ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

scholarly journals Studies on gastric mucoproteins. The isolation and characterization of the mucoprotein of the water-soluble mucus from pig cardiac gastric mucosa

1971 ◽  
Vol 123 (5) ◽  
pp. 845-853 ◽  
Author(s):  
D. Snary ◽  
A. Allen
Keyword(s):  
Sialic Acid ◽  
Gastric Mucosa ◽  
Gel Filtration ◽  
Water Soluble ◽  
Group Activities ◽  
Isolation And Characterization ◽  
Two Peaks ◽  
Sepharose 4B

1. Gel filtration of the water-soluble radioactive mucus produced three radioactive fractions, fraction A excluded on Sepharose 4B, fraction B included on Sepharose 4B but excluded on Sephadex G-200, and fraction C included on Sephadex G-200. 2. The specific radioactivities of fractions A and B were the same, with fraction C a little lower, whether the material was labelled with 14C-labelled carbohydrate or with 3H-labelled protein prepared by incubation of mucosal scrapings in vitro with [U-14C]glucose or [G-3H]threonine respectively. 3. Fractions A and B had an analysis of protein 22%, hexose 28%, hexosamine 28%, fucose 10% and sialic acid 1%; fraction C had an analysis closely similar to this, except that it contained about 10% of a protein contaminant. 4. All three fractions had closely similar A and H blood-group activities. 5. Ultracentrifuge studies showed fractions A, B and C were polydisperse with s025,w values of 18.7S, 4.9S and 3.9S respectively. 6. The unfractionated water-soluble mucus contained only two peaks, fraction A 18.7S and a peak of 4.4S, which was a combination of fractions B and C. 7. The radioactive mucoprotein accounted for 85% by weight of the soluble mucus and the results show that it consisted of two distinct fractions A and B–C, which were chemically, biosynthetically and immunologically very similar.

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