scholarly journals Isolation and characterization of the CNBr peptides from the proteolytically derived N-terminal fragment of ovine opsin

1983 ◽  
Vol 211 (3) ◽  
pp. 661-670 ◽  
Author(s):  
M Brett ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Gel Permeation Chromatography ◽  
Isolation And Characterization ◽  
Peptide Mixture ◽  
Gel Permeation ◽  
Membrane Bound ◽  
Sepharose 4B ◽  
Differential Solubility

Ovine rhodopsin may be cleaved in situ by Staphylococcus aureus V8 proteinase into two membrane-bound fragments designated V8-L (27 000 mol.wt.) and V8-S (12 000 mol.wt.). After purification of the proteolysed complex by affinity chromatography in detergent using concanavalin A immobilized on Sepharose 4B, the two polypeptide fragments may be separated by gel-permeation chromatography on Sephadex LH-60. Digestion of the N-terminal-derived V8-L fragment with CNBr in 70% (v/v) trifluoroacetic acid resulted in a peptide mixture that could be fractionated by procedures involving gel-permeation chromatography in organic and aqueous solvents and the use of differential solubility. The complete or partial sequences of all ten peptides are reported.

scholarly journals Sequence variability in the retinal-attachment domain of mammalian rhodopsins

1984 ◽  
Vol 217 (3) ◽  
pp. 605-613 ◽  
Author(s):  
D J C Pappin ◽  
J B C Findlay
Keyword(s):  
Staphylococcus Aureus ◽  
Visual Pigment ◽  
Gel Filtration ◽  
Aldehyde Group ◽  
Marked Variation ◽  
Sequence Variability ◽  
Membrane Bound ◽  
Covalently Linked ◽  
Sepharose 4B

Ovine rhodopsin was regenerated with 11-cis-[15-3H]retinal and cleaved in situ by Staphylococcus aureus V8 proteinase to give two membrane-bound fragments of Mr 27 000 (V8-L) and 12 000 (V8-S). After purification of the proteolysed complex by affinity chromatography with concanavalin A-Sepharose 4B, [3H]retinal was covalently linked to the protein by reduction with borohydride. The purified [3H]-retinyl V8-S fragment was cleaved with CNBr and trifluoroacetic acid, the resulting peptides resolved by gel filtration and the [3H]retinyl peptide sequenced. The protocol developed for the isolation and sequencing of this region of the ovine protein was applied directly, and reproducibly, to bleached and unregenerated porcine and equine opsins. Comparisons of the primary structures of the fragments reveals marked variation in the sequence immediately after the lysine residue shown in the ovine protein to be the attachment point for the aldehyde group of the chromophore. Mutable positions are localized in regions previously predicted as adopting nonregular or distorted conformations and hint at structural arrangements that may provide a better understanding of the spectral and functional properties of the visual pigment.

scholarly journals Molar mass determination of lignins and characterization of their polymeric structures by multi-detector gel permeation chromatography

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Evamaria C. Gaugler ◽  
Wolfgang Radke ◽  
Andrew P. Vogt ◽  
Dawn A. Smith
Keyword(s):  
Molar Mass ◽  
Lithium Bromide ◽  
Gel Permeation Chromatography ◽  
Mass Determination ◽  
Organosolv Lignin ◽  
Gel Permeation ◽  
Softwood Kraft Lignin ◽  
Polymeric Structures

AbstractMolar masses, Mark-Houwink-Sakurada (MHS) exponents, and refractive index increments (dn/dc) for three lignins were determined without derivatization by multi-detector gel permeation chromatography (GPC) in dimethylformamide (DMF) with 0.05 M lithium bromide (LiBr). The lack of effectiveness of fluorescence filters on molar mass determination by GPC-multi-angle laser light scattering (MALS) was confirmed for softwood kraft lignin (Indulin AT) and revealed for mixed hardwood organosolv lignin (Alcell) as well as soda straw/grass lignin (Protobind 1000). GPC with viscometry detection confirmed that these lignins were present as compact molecules. The MHS exponent α for Indulin AT and Alcell was in the order of 0.1. Additionally, the intrinsic viscosity of Protobind 1000 for a given molar mass was much lower than that of either Alcell or Indulin AT. This is the first report of dn/dc values for these three lignins in DMF with 0.05 M LiBr.

scholarly journals Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement

1992 ◽  
Vol 284 (1) ◽  
pp. 169-176 ◽  
Author(s):  
T R Hughes ◽  
S J Piddlesden ◽  
J D Williams ◽  
R A Harrison ◽  
B P Morgan
Keyword(s):  
Affinity Purification ◽  
Membrane Attack Complex ◽  
Erythrocyte Membranes ◽  
Dependent Manner ◽  
Inhibitory Protein ◽  
Rat Erythrocytes ◽  
Butanol Extract ◽  
Isolation And Characterization ◽  
Membrane Bound

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated ‘reactive’ lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.

Synthesis and chromatographic characterization of [Tc-99m]technetium-humic acid species

2000 ◽  
Vol 88 (2) ◽  
Author(s):  
D. Rößler ◽  
K. Franke ◽  
R. Süß ◽  
E. Becker ◽  
H. Kupsch
Keyword(s):  
Humic Acid ◽  
Stannous Chloride ◽  
Gel Permeation Chromatography ◽  
Paper Electrophoresis ◽  
Gel Permeation ◽  
Soil Humic Acid ◽  
Chromatographic Characterization ◽  
Ph Dependent ◽  
Layer Chromatography

A natural moor soil humic acid (HA) was labeled with Tc-99m via reduction of pertechnetate with stannous chloride. The humic acid species obtained were characterized by thin layer chromatography (TLC), gel permeation chromatography (GPC), sequential chromatographic analysis (SCA), paper electrophoresis and micropore filtration. Labeling was found to take place in all ranges of molecular weight. Due to the complex humic acid composition and the formation of hydroxo species the labeling yields strongly depend on the separation conditions, ranging from 42% to 80%. The pH-dependent distribution of mobile and immobile species was determined by SCA for HTcO

Kinetic Studies on the Curing Reactions of Fluoroepoxy Oligomer and Cycloaliphatic Amine

2013 ◽  
Vol 747 ◽  
pp. 753-756 ◽  
Author(s):  
Thitinun Chongtum ◽  
Wunpen Chonkaew
Keyword(s):  
Average Molecular Weight ◽  
Gel Permeation Chromatography ◽  
Kinetic Studies ◽  
Equivalent Weight ◽  
Number Average Molecular Weight ◽  
Polymeric Systems ◽  
Acid Titration ◽  
Curing Kinetic ◽  
Gel Permeation

The curing kinetic analysis is an important technique for the characterization of the curing behavior of reactive polymeric systems. In this study, fluoroepoxy oligomer was synthesized from trifluoromethyl aniline and epichlorohydrin. The epoxide equivalent weight (EEW) and the number average molecular weight (Mn) of the systhesized fluroepoxy oligomer determined from acid titration and gel permeation chromatography were found to be 312.16 g/eq and 534 g/mol, respectively. The mixtures of the fluoroepoxy oligomer were mixed with the cycloaliphatic amine in various stiochiometric ratios (1:1, 1: 1.5 and 1:2). The effects of the stiochiometric ratio on the curing behaviors were studied using both isothermal and non-isothermal DSC methods. Ozawas, Kissingers and Friedmans methods were employed to investigate the kinetic parameters. The results showed that the peak temperature (Tp) increased with the increasing heating rate. The activation energy (Ea) calculated from Ozawas and Kissingers methods were much larger than that from Friedmans method.

scholarly journals Elemental and spectroscopic characterization of humic acids fractionated by gel permeation chromatography

Agronomie ◽  
2000 ◽  
Vol 20 (5) ◽  
pp. 491-504 ◽  
Author(s):  
Aaziz Ouatmane ◽  
Valeria Dorazio ◽  
Mohamed Hafidi ◽  
Jean-Claude Revel ◽  
Nicola Senesi
Keyword(s):  
Humic Acids ◽  
Gel Permeation Chromatography ◽  
Spectroscopic Characterization ◽  
Gel Permeation

Soluble neutral maltase–glucoamylase from the small intestine: separation and characterization of components with differing affinity for concanavalin A

1982 ◽  
Vol 60 (11) ◽  
pp. 1007-1013 ◽  
Author(s):  
G. Forstner ◽  
A. Salvatore ◽  
L. Lee ◽  
J. Forstner
Keyword(s):  
Concanavalin A ◽  
Gradient Elution ◽  
Soluble Form ◽  
Rat Intestine ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Neutral Ph ◽  
Membrane Bound ◽  
Sepharose 4B

Intestinal maltase with a neutral pH optimum exists in both a brush border membrane-bound form and a soluble form in suckling rat intestine. Previous experiments in our laboratory have shown that the soluble enzyme contains a component which binds much more tightly to concanavalin A (ConA) than solubilized forms of the membrane enzyme. We studied the origin of this component by subjecting neutral, soluble maltase activity to chromatography on Sepharose 4B at age 13, 18 (preweaning), and 25 (postweaning) days. At 13 days, two maltase peaks were obtained with approximate molecular weights of 400 000 (peak I) and 150 000 (peak II). Peak II was less prominent at 18 days and was absent at 25 days. At 13 days, the majority of peak I consisted of material which was bound between 0.025 and 0.05 M α-methyl mannoside on gradient elution chromatography of ConA-Sepharose. Peak II contained material which eluted between 0.075 and 0.3 M α-methyl mannoside. At 25 days, all of the soluble maltase eluted between 0.025 and 0.04 M α-methyl mannoside. Peak I and peak II maltases had similar pH optima and Km's for maltase. Peak II maltase had a fourfold greater activity toward glycogen than peak I maltase with approximately the same activity for palatinose, turanose, and trehalose. Both maltases were precipitated by an antibody raised against adult membrane-bound maltase. Soluble maltase with neutral pH activity in the suckling rat intestine, therefore, consists of two immunologically related isozymes which differ in their molecular weight, their binding by ConA, and their specificity for glycogen. The small isozyme disappears at or about the time of weaning.

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