Determination of Absolute Molecular Weights Using Sedimentation Equilibrium Analytical Ultracentrifugation

Author(s):  
Stephen E. Harding
1977 ◽  
Vol 81 (2) ◽  
pp. 447-449 ◽  
Author(s):  
Riva Rubinstein ◽  
Anthony C.H. Durham

2003 ◽  
Vol 185 (5) ◽  
pp. 1693-1700 ◽  
Author(s):  
Li Zhao ◽  
Shuji Kanamaru ◽  
Chatree'chalerm Chaidirek ◽  
Fumio Arisaka

ABSTRACT Two proteins, gp15 and gp3 (gp for gene product), are required to complete the assembly of the T4 tail. gp15 forms the connector which enables the tail to bind to the head, whereas gp3 is involved in terminating the elongation of the tail tube. In this work, genes 15 and 3 were cloned and overexpressed, and the purified gene products were studied by analytical ultracentrifugation, electron microscopy, and circular dichroism. Determination of oligomerization state by sedimentation equilibrium revealed that both gp15 and gp3 are hexamers of the respective polypeptide chains. Electron microscopy of the negatively stained P15 and P3 (P denotes the oligomeric state of the gene product) revealed that both proteins form hexameric rings, the diameter of which is close to that of the tail tube. The differential roles between gp15 and gp3 upon completion of the tail are discussed.


2020 ◽  
Vol 49 (8) ◽  
pp. 781-789 ◽  
Author(s):  
Jennifer M. K. Wakefield ◽  
Susan Braovac ◽  
Hartmut Kutzke ◽  
Robert A. Stockman ◽  
Stephen E. Harding

AbstractThe Oseberg ship is one of the most important archaeological testimonies of the Vikings. After excavation in 1904, the wooden gravegoods were conserved using alum salts. This resulted in extreme degradation of a number of the objects a hundred years later through acid depolymerisation of cellulose and lignin. The fragile condition of the artefacts requires a reconsolidation which has to be done avoiding water as solvent. We synthesized tert-butyldimethylsilyl (TBDMS) chitosan which is soluble in a 50:50 solution of ethyl acetate and toluene. Measurement of its molecular weight, to anticipate its penetration, provided a challenge as the density difference of the polymer and solvent was too small to provide adequate solute redistribution under a centrifugal field, so a two-stage process was implemented (i) determination of the weight-average molar mass of the aqueous soluble activated precursor, chitosan mesylate, Mw,mc using sedimentation equilibrium with the SEDFIT-MSTAR algorithm, and determination of the degree of polymerisation DP; (ii) measurement of the average degree of substitution DSTBDMS of the TBDMS group on each chitosan monosaccharide monomer unit using NMR, to augment the Mw,mc value to give the molar mass of the TBDMS-chitosan. For the preparation, we find Mw = 9.8 kg·mol−1, which is within the acceptable limit for penetration and consolidation of degraded wood. Future work will test this on archaeological wood from different sources.


In a previous paper, the investigation of the scattering of light in agar sols and gels was described and a view regarding the changes taking place in the system during gelation was developed. In a series of paper, of which this is the first, the author proposes to publish investigations of the scattering of light in protein solutions. The various physical properties of the different proteins have been studied for a long time past. Several workers have tried to evaluate the molecular weights of the proteins from the osmotic pressure of their solutions and also from analytical data. Recently a very precise and definite method for the determination of the molecular weights of the proteins, based upon the sedimentation of these heavy molecules in the ultra-centrifuge, has been successfully developed by Svedberg. The molecular weight can be determined in two ways:—(I) by the measurement of the sedimentation equilibrium reached in the cell as a result of the centrifugal and diffusion forces; (II) by measuring the sedimentation velocity of the protein molecules in high centrifugal fields.


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