Molecular-weight determination by gel chromatography

The highest degree of purification was achieved with extracellular endopectate lyase from the anaerobic bacterium Clostridium fel-sineum.Mi A 225-fold purified preparation, homogeneous in disc electrophoresis, was obtained by precipitation with ethanol, and chromatography on CM-cellulose and on Sephadex G-200. Its molecular weight (determined by gel chromatography) was 105,000. 

The dextran used in this work was the commercially available Dextran T-40 (Farmacia), produced by Leuconostoc mesenteroides B512 species. The average molecular weight determined by gel permeation chromatography was = 13,000, = 36,000. Dex-... 

Spatorico, A.L. and Coulter, B. (1973) Molecular-weight determinations by gel-permeation chromatography and viscometry. Journal of Polymer Science Part B-Polymer Physics, 11,1139-1150. 

Solubility tests carried out on the polymers revealed that all the LC poly (azomethine esters) were insoluble in common organic solvents such as chloroform, THF and acetone. This leads to the difficulty in their molecular weight determination by gel permeation chromatography (GPC). The inherent viscosities (r ii ) of the polymers 3 (a-d) were as given in Table 12.1. The low inherent viscosities may be attributed to the poor solubility of the polymers in the polymerization solution. Precipitate was also formed during the polymerization which hinders further propagation of the polymer chain and thus leads to lower molecular weight. 

The molecular weight determined by the gel filtration chromatography with Toyopearl 55 HW was 115 kdal while that estimated by SDS-PAGE was 43 kdal (Figure 4,5). 

Since the actual structures of the pores of the insoluble polymer supports used as gel permeation chromatography resins are not known, an exact theoretical treatment of the technique is not possible, and the gel permeation characteristics of a solute can only be compared in a relative fashion. (In alluding to molecular weights determined by this method, we shall use the term Mapp since values obtained by this method are approximate.) All estimates of polymer dimensions based on gel chromatography are therefore relative and based on those determined for polymers whose dimensions are known. 

Figure 4. Molecular weight determination by analytical gel chromatography under nondenaturing conditions. Plot of the cube root of the distribution coefficient versus the cube root of the molecular weight (27).

Three histone-specific acetyltransferases have been partially purified and characterized from rat thymus nuclei (225). The enzymes were extracted from rat thymus nuclei by sonication in the presence of 1M ammonium sulfate and separated into two active fractions (A and B) by DEAE-cellulose chromatography. Fraction B was further separated into two active fractions (Bi and B2) by gel filtration on Sephadex G-200. Each fraction was then purified further by chromatography on hydroxyapatite. The molecular weights, determined by Sephadex G-200 and by sucrose density gradient centrifugation, were 99,000, 110,000, and 92,000 for enzymes A, Bi, and B2, respectively. All three enzymes required acetyl CoA as acetate donor, and the activity of the enzymes was inhibited by p-chloromercuribenzoate. Acetyltransferase A preferentially acetylated histone I (FI) and also poly-L-lysine. Acetyltransferase Bi and B2 preferred histone H4 (other names IV, F2al) and did not acet-ylate poly-L-lysine and histone H3 (III, F3). In addition to c-N-acetyl-lysine, two other unidentified amino acid derivatives were obtained from a digest of histone H4 acetylated by the two B enzymes. 

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