scholarly journals Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds

2011 ◽  
Vol 2011 ◽  
pp. 1-5 ◽  
Author(s):  
Shannen Lau ◽  
Bim Graham ◽  
Ben J. Boyd ◽  
Colin W. Pouton ◽  
Paul J. White
Keyword(s):  
Serum Albumin ◽  
Ammonium Acetate ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Purification Process ◽  
Ammonium Acetate Buffer ◽  
Commercial Supplier ◽  
Potential Benefits ◽  
Maleimide Group ◽  
Phosphate Buffered Saline

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions.

LC–MS/MS Determination of Apigenin in Rat Plasma and Application to Pharmacokinetic Study

2020 ◽  
Vol 21 ◽  
Author(s):  
Shixing Zhu ◽  
Jiayuan Zhang ◽  
Zhihua Lv ◽  
Mingming Yu
Keyword(s):  
Formic Acid ◽  
Pharmacokinetic Study ◽  
Ammonium Acetate ◽  
Rat Plasma ◽  
Biological Properties ◽  
Plasma Samples ◽  
Supply Rate ◽  
Natural Plant ◽  
Ammonium Acetate Buffer

Background: Apigenin, a natural plant flavone, has been shown to possess a variety of biological properties. Objective: In this report, a highly selective and sensitive LC-MS/MS method was developed and validated for the determination of apigenin in rat plasma. Methods: Analysts were separated on the HSS T3 column (1.8 μm 2.1×100 mm) using acetonitrile and 0.1% formic acid in 2 mM ammonium acetate buffer at a supply rate of 0.200 mL/min as eluent in gradient model. Results: Plasma samples were treated by protein precipitation using acetonitrile for the recovery ranging from 86.5% to 90.1% for apigenin. The calibration curves followed linearity in the concentration range of 0.50-500 ng/mL. The inter-day and intra-day precisions at different QC levels within 13.1% and the accuracies ranged from -10.6% to 8.6%. Conclusion: The assay has been successfully applied to the pharmacokinetic study of apigenin in rats.

scholarly journals Serum Albumin Redox States: More than Oxidative Stress Biomarker

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 503
Author(s):  
Fuka Tabata ◽  
Yasuaki Wada ◽  
Satomi Kawakami ◽  
Kazuhiro Miyaji
Keyword(s):  
Oxidative Stress ◽  
Serum Albumin ◽  
Animal Studies ◽  
Redox State ◽  
Ex Vivo ◽  
Pathological Condition ◽  
Analytical Techniques ◽  
Cysteine Residue ◽  
Research Field ◽  
Systemic Oxidative Stress

Serum albumin is the most abundant circulating protein in mammals including humans. It has three isoforms according to the redox state of the free cysteine residue at position 34, named as mercaptalbumin (reduced albumin), non-mercaptalbumin-1 and -2 (oxidized albumin), respectively. The serum albumin redox state has long been viewed as a biomarker of systemic oxidative stress, as the redox state shifts to a more oxidized state in response to the severity of the pathological condition in various diseases such as liver diseases and renal failures. However, recent ex vivo studies revealed oxidized albumin per se could aggravate the pathological conditions. Furthermore, the possibility of the serum albumin redox state as a sensitive protein nutrition biomarker has also been demonstrated in a series of animal studies. A paradigm shift is thus ongoing in the research field of the serum albumin. This article provides an updated overview of analytical techniques for serum albumin redox state and its association with human health, focusing on recent findings.

Polarographic Determination of Zinc in Compounded Rubber

1950 ◽  
Vol 23 (4) ◽  
pp. 975-980
Author(s):  
F. C. J. Poulton ◽  
L. Tarrant
Keyword(s):  
Zinc Oxide ◽  
Ammonium Acetate ◽  
Potassium Thiocyanate ◽  
Acetate Buffer Solution ◽  
Acid Electrolyte ◽  
Buffer Solution ◽  
Ammonium Acetate Buffer ◽  
Ammonium Acetate Buffer Solution ◽  
Gravimetric Procedure

Abstract Reasons are advanced for the unsatisfactory nature of some of the older methods for the determination of very small amounts of zinc in compounded rubber, particularly in latex mixings. The polarographic technique offers a possible solution, but most of the commoner electrolytes for the electroreduction of this metal are alkaline, and give rise to similar errors as are met in the gravimetric procedure. The development of a suitable acid electrolyte was therefore undertaken, and ways of dealing with likely interferences were examined. The electroltye finally recommended is a potassium thiocyanate-ammonium acetate buffer solution; iron, when present, is reduced to the ferrous condition by potassium iodide. The method was used to determine zinc oxide in a series of mixings of known composition ranging from 0.8 to 40 per cent. In all except the highest proportions of zinc oxide, the figures obtained agree well with the theoretical.

scholarly journals Production and comparison of mature single-domain ‘trefoil’ peptides pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58

1995 ◽  
Vol 308 (3) ◽  
pp. 1001-1007 ◽  
Author(s):  
M P Chadwick ◽  
F E B May ◽  
B R Westley
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Gel Filtration ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Exchange Chromatography ◽  
Single Domain ◽  
Trefoil Peptides ◽  
Trefoil Peptide

The preparation and purification of recombinant mature pNR-2/pS2, a single-domain member of the ‘trefoil’ family of cysteine-rich secreted proteins, is described. Analysis of recombinant pNR-2/pS2 by ion-exchange chromatography showed that it was heterogeneous. The heterogeneity was reduced by treatment with thiol-group-containing reagents, suggesting that it is caused by the odd number of cysteine residues in mature pNR-2/pS2, and this view was reinforced by mutation of the extra-trefoil domain cysteine residue, Cys58, to a serine residue. Electrophoresis of recombinant pNR-2/pS2 Cys58 and pNR-2/pS2 Ser58 proteins under non-denaturing conditions confirmed that the Ser58 mutant is much more homogeneous, and showed that most of pNR-2/pS2 Ser58 co-migrates as a single band with pNR-2/pS2 secreted from breast-cancer cells in culture. Treatment of recombinant pNR-2/pS2 proteins with various thiol-group-reactive reagents indicated that cysteine is the most effective at producing recombinant pNR-2/pS2 that co-migrates with pNR-2/pS2 secreted by breast-cancer cells. Dithiothreitol appeared to denature the proteins, and GSH was relatively ineffective. pNR-2/pS2 Cys58 treated with cysteine and untreated pNR-2/pS2 Ser58 had the same apparent molecular mass, measured by gel filtration, as pNR-2/pS2 secreted from breast-cancer cells. This is the first report of the production of a recombinant mature single-domain trefoil peptide and should greatly facilitate elucidation of the structure and function of pNR-2/pS2.

scholarly journals High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection

1986 ◽  
Vol 234 (3) ◽  
pp. 629-633 ◽  
Author(s):  
C K Lim ◽  
F Li ◽  
T J Peters
Keyword(s):  
Mobile Phase ◽  
High Performance ◽  
Ammonium Acetate ◽  
Amperometric Detection ◽  
Reversed Phase ◽  
Fluorescent Detection ◽  
Organic Modifiers ◽  
Ammonium Acetate Buffer ◽  
Effects Of Ph ◽  
Ph Buffer

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.

Calcium ion binding to clinically relevant chemical modifications of human serum albumin

1995 ◽  
Vol 41 (11) ◽  
pp. 1654-1661 ◽  
Author(s):  
H Vorum ◽  
K Fisker ◽  
M Otagiri ◽  
A O Pedersen ◽  
U Kragh-Hansen
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Conformational Changes ◽  
Calcium Binding ◽  
Equilibrium Dialysis ◽  
Thiol Group ◽  
Protein Molecule ◽  
Penicillin G ◽  
Lysine Residues

Abstract Calcium binding to glycated, penicilloylated, acetylated, and normal defatted human serum albumin as well as to mercapt- and nonmercaptalbumin was studied by equilibrium dialysis of radioactive Ca2+. Binding was quantified by five Scatchard constants [ni = 1, (i = 1-4) and n5 = 10]. Glycation resulted in increased k1- and k2-values and unchanged k3-k5-values, whereas penicilloylation increased all five association constants. The increments were greater the more pronounced the modification, and the enhancements caused by penicilloylation were, for the same degree of modification, greater than those produced by glycation. In contrast, acetylation by acetylsalicylate did not affect calcium binding. Likewise, binding to mercapt- and nonmercaptalbumin was the same, a finding showing that the thiol group of cysteine 34 is not important for calcium binding. D-Glucose and penicillin G are known to react with lysine residues of albumin, and the enhancement of binding resulting from glycation or penicilloylation is probably brought about by unspecific electrostatic effects, possibly supplemented by conformational changes of the protein molecule. The relative importance of the three domains of human serum albumin for calcium binding is discussed.

scholarly journals Iodination of glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus

1976 ◽  
Vol 155 (3) ◽  
pp. 523-534 ◽  
Author(s):  
G Allen ◽  
J I Harris
Keyword(s):  
Conformational Changes ◽  
Bacillus Stearothermophilus ◽  
Three Dimensional ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Enzymic Activity ◽  
Three Dimensional Model ◽  
Muscle Enzyme ◽  
Tyrosine Residues ◽  
Pig Muscle

The reaction of iodine with glyceraldehyde 3-phosphate dehydrogenase from Bacillus stearothermophilus was investigated. The active-site thiol group of the cysteine residue homologous with cysteine-149 in the pig muscle enzyme was protected by reaction with tetrathionate. The apoenzyme was readily inhibited by KI3 solution at pH8, but the coenzyme, NAD+, protected the enzyme against inhibition and decreased the extent of iodination. At pH 9.5, ready inhibition of both apo- and holo-enzyme was observed. Tryptic peptides containing residues iodinated at pH 8 were isolated and characterized. One of the most reactive residues in both holo- and apo-enzymes was a tyrosine homologous with tyrosine-46 in the pig muscle enzyme, and this residue was iodinated without loss of enzymic activity. Other reactive tyrosine residues in the apoenzyme were in positions homologous with residues 178, 273, 283 and 311 in the pig muscle enzyme, but they were not readily iodinated in the holoenzyme. Histidine residues in both holo- and apo-enzymes were iodinated at pH 8 in sequence positions homologous with residues 50, 162 and 190 in the pig muscle enzyme. The inhibition of the enzyme was not correlated with the iodination of a particular residue. The results are discussed in relation to a three-dimensional model based on the structure of the lobster muscle enzyme and demonstrate that conformational changes affecting the reactivity of several tyrosine residues most probably occur on binding of the coenzyme.

scholarly journals Oligolysine-based saccharide clusters: synthesis and specificity

2002 ◽  
Vol 368 (1) ◽  
pp. 111-119 ◽  
Author(s):  
Natacha FRISON ◽  
Philippe MARCEAU ◽  
Annie-Claude ROCHE ◽  
Michel MONSIGNY ◽  
Roger MAYER
Keyword(s):  
Plasma Membrane ◽  
Hepatoma Cell ◽  
Surface Membrane ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Cell Uptake ◽  
Amide Bond ◽  
Hepatoma Cell Line ◽  
Human Hepatoma Cell ◽  
Plant Lectins

In search of specific and highly selective sugar clusters for cell receptors, such as membrane lectins, various disaccharides were coupled to small peptide cores through an amide bond. In a first step, the reducing disaccharides, i.e. lactose and three different dimannoses, were converted into glycosyl-pyroglutamyl-β-alanine derivatives. The free carboxylic group of these conjugates was then coupled to the α and ∊ amino groups of the core peptide (Lysn-Ala-Cys-NH2) with n = 1 to 5, with complete substitution leading to homogeneous glycoclusters. The thiol group of the cysteine residue was used to tag the glycosylated oligolysines upon reaction with fluorescein iodoacetamide. The affinity of these glycoclusters towards two plant lectins was assessed by surface plasmon resonance. The selectivity of their cell uptake was investigated by flow cytometry using two types of cells: a human hepatoma cell line (HepG2 cells) expressing the plasma membrane galactose-specific lectin, and monocyte-derived dendritic cells expressing the plasma membrane mannose-specific lectin. The glycoclusters containing four or five disaccharides were shown to bind plant lectins and cell surface membrane lectins with a narrow selectivity and with a high affinity.

The Separation of two Deoxyribonucleases from Extracts of Intestinal Mucosa of the Rat

1972 ◽  
Vol 50 (6) ◽  
pp. 697-703 ◽  
Author(s):  
C. Y. Lee ◽  
Sheilia Lawrence ◽  
S. H. Zbarsky
Keyword(s):  
Intestinal Mucosa ◽  
Ammonium Acetate ◽  
Divalent Cations ◽  
Deae Cellulose ◽  
Sodium Formate ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dnase Ii ◽  
Ammonium Acetate Buffer ◽  
Optimum Activity

Two deoxyribonucleases have been demonstrated in cell-free extracts of rat intestinal mucosa. The enzymes were separated by ion-exchange chromatography on DEAE-cellulose and further purified by partition on hydroxyapatite. One DNase had optimum activity at pH 6.8–7.0 in ammonium acetate buffer, required Mn2+ or Mg2+ for activity, and was inhibited by EDTA. This enzyme is a DNase I by Laskowski's criteria Advan. Enzymol. 29, 165 (1967). The second enzyme was a DNase II with optimum activity at pH 3.5–4.0 in sodium formate buffer, was not inhibited by EDTA, and showed no requirement for divalent cations. Both enzymes were active only with native DNA and had no action on heated DNA or purified yeast transfer RNA.

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