A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development

1997 ◽  
Vol 25 (5-6) ◽  
pp. 437-443 ◽  
Author(s):  
P. G. Varley ◽  
A. J. Brown ◽  
H. C. Dawkes ◽  
N. R. Burns
Keyword(s):  
Analytical Ultracentrifugation ◽  
Sedimentation Equilibrium

scholarly journals Terpene polyacrylate TPA5 shows favorable molecular hydrodynamic properties as a potential bioinspired archaeological wood consolidant

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Michelle Cutajar ◽  
Fabrizio Andriulo ◽  
Megan R. Thomsett ◽  
Jonathan C. Moore ◽  
Benoit Couturaud ◽  
...  
Keyword(s):  
Molar Mass ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Axial Ratio ◽  
Sedimentation Equilibrium ◽  
Archaeological Wood ◽  
Molecular Hydrodynamic ◽  
Pine Trees ◽  
Minimal Concentration ◽  
Hydrogen Bonding Potential

AbstractThere is currently a pressing need for the development of novel bioinspired consolidants for waterlogged, archaeological wood. Bioinspired materials possess many advantages, such as biocompatibility and sustainability, which makes them ideal to use in this capacity. Based on this, a polyhydroxylated monomer was synthesised from α-pinene, a sustainable terpene feedstock derived from pine trees, and used to prepare a low molar mass polymer TPA5 through free radical polymerisation. This polymer was extensively characterised by NMR spectroscopy (chemical composition) and molecular hydrodynamics, primarily using analytical ultracentrifugation reinforced by gel filtration chromatography and viscometry, in order to investigate whether it would be suitable for wood consolidation purposes. Sedimentation equilibrium indicated a weight average molar mass Mw of (4.3 ± 0.2) kDa, with minimal concentration dependence. Further analysis with MULTISIG revealed a broad distribution of molar masses and this heterogeneity was further confirmed by sedimentation velocity. Conformation analyses with the Perrin P and viscosity increment ν universal hydrodynamic parameters indicated that the polymer had an elongated shape, with both factors giving consistent results and a consensus axial ratio of ~ 4.5. These collective properties—hydrogen bonding potential enhanced by an elongated shape, together with a small injectable molar mass—suggest this polymer is worthy of further consideration as a potential consolidant.

Biophysical Characterization of Interaction between E. coli Alanyl-tRNA Synethase with its Promoter DNA

2020 ◽  
Vol 27 (7) ◽  
pp. 635-648
Author(s):  
Baisakhi Banerjee ◽  
Sayak Ganguli ◽  
Rajat Banerjee
Keyword(s):  
Dna Binding ◽  
Analytical Ultracentrifugation ◽  
Isothermal Calorimetry ◽  
Trna Synthetase ◽  
Full Length ◽  
Sedimentation Equilibrium ◽  
Titration Calorimetry ◽  
E Coli ◽  
Promoter Dna

Background: Aminoacyl-tRNA Synthetases (aaRSs) are well known for their role in the translation process. Lately investigators have discovered that this family of enzymes are also capable of executing a broad repertoire of functions that not only impact protein synthesis, but extend to a number of other activities. Till date, transcriptional regulation has so far only been described in E. coli Alanyl-tRNA synthetase and it was demonstrated that alaRS binds specifically to the palindromic DNA sequence flanking the gene’s transcriptional start site and thereby regulating its own transcription. Objective: In the present study, we have characterized some of the features of the alaRS-DNA binding using various biophysical techniques. Methods: To understand the role of full length protein and oligomerization of alaRS in promoter DNA binding, two mutants were constructed, namely, N700 (a monomer, containing the N-terminal aminoacylation domain but without the C-terminal part) and G674D (previously demonstrated to form full-length monomer). Protein-DNA binding study using fluorescence spectroscopy, analytical ultracentrifugation, Isothermal Titration Calorimetry was conducted. Results: Sedimentation equilibrium studies clearly demonstrated that monomeric variants were unable to bind promoter DNA. Isothermal Calorimetry (ITC) experiment was employed for further characterization of wild type alaRS-DNA interaction. It was observed that full length E. coli Alanyl-tRNA synthetase binds specifically with its promoter DNA and forms a dimer of dimers. On the other hand the two mutant variants were unable to bind with the DNA. Conclusion: In this study it was concluded that full length E. coli Alanyl-tRNA synthetase undergoes a conformational change in presence of its promoter DNA leading to formation of higher order structures. However, the exact mechanism behind this binding is currently unknown and beyond the scope of this study.

scholarly journals Purification and characterization of goblet-cell mucin of high Mr from the small intestine of sheep

1985 ◽  
Vol 229 (2) ◽  
pp. 419-428 ◽  
Author(s):  
T K S Mukkur ◽  
D L Watson ◽  
K S Saini ◽  
A K Lascelles
Keyword(s):  
Small Intestine ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Goblet Cells ◽  
Immunofluorescence Assay ◽  
Equilibrium Analysis ◽  
Sedimentation Equilibrium ◽  
Equilibrium Density ◽  
Total Carbohydrate ◽  
Protamine Sulphate

Crude soluble mucus from sheep small intestine was freed of nearly all the nucleic acid contaminants by precipitation with protamine sulphate and treatment with nucleases. After removal of non-covalently bound proteins by equilibrium density-gradient centrifugation in CsCl, a high-Mr glycoprotein was isolated by repeated h.p.l.c. from the partially purified mucin. The high degree of purity of the high-Mr mucin was borne out by (a) the observation of a single boundary on analytical ultracentrifugation in the presence of 5M-guanidinium chloride and (b) the observation of apparent monodispersity on sedimentation-equilibrium analysis. The Mr of the highly purified mucin, determined by sedimentation equilibrium, was 5.0 (+/- 0.1) X 10(6) and was concentration-independent. Finally, only goblet cells and the mucus blanket lining the intestinal epithelial cells were immunofluorescent when guinea-pig anti-(highly purified mucin) serum was used in an indirect immunofluorescence assay. The above antiserum reacted with apparently equal strength with goblet cells and with free mucin in abomasum, caecum and colon. The chemical composition of the glycoprotein was 66% carbohydrate and 34% protein, 45% of the latter being composed of valine and threonine. The glycoprotein migrated anodally on immunoelectrophoresis and contained 7.1% (w/w) sulphate. Neutral hexoses accounted for nearly half of the total carbohydrate content, followed by galactosamine and glucosamine. Whereas fucose and sialic acid were present in only small amounts, uronic acid was not detectable in the highly purified mucus glycoprotein.

scholarly journals Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

2015 ◽  
Vol 109 (4) ◽  
pp. 827-837 ◽  
Author(s):  
Jia Ma ◽  
Michael Metrick ◽  
Rodolfo Ghirlando ◽  
Huaying Zhao ◽  
Peter Schuck
Keyword(s):  
Analytical Ultracentrifugation ◽  
Sedimentation Equilibrium ◽  
Variable Field
Sign in / Sign up

Export Citation Format

Share Document