Peptide purification by gel filtration in aqueous pyridine--ammonia.

A method is described for the purification of peptides by gel filtration on Sephadex. The success of the method is due mainly to the use of 70% ($\text{v}\text{v}$) pyridine-30% ($\text{v}\text{v}$) 1 m aqueous ammonia, an excellent volatile solvent for peptides which does not degrade Sephadex. The method has been used to purify all of the major peptides of cowpea chlorotic mottle virus coat protein, after an initial fractionation by ion-exchange chromatography, and selected separations are used to illustrate the degree of fractionation which can be achieved.     

Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.     

The separation of microsomal membranes and free polyribosomes by gel filtration.

The integrity of polyribosome-membrane complexes in microsomal preparations is dependent on the centrifugal conditions used for their preparation. This paper describes techniques based on gel filtration that enable the rapid and gentle separation of microsomal membranes from both free polyribosomes and soluble protein.     

A new assay of estrogen receptor by thin-layer gel filtration.

Application of a thin-layer gel filtration (TLG) method using Sephadex G-200 superfine for the assay of estrogen receptor (ER) is described. The method is based on the effective separation of specific receptor protein from nonspecific binding proteins and free hormone by TLG and subsequent determination of the radioactivity bound to specific ER. By the aid of fluorescein-labeled human γ-globulin, the position of specific ER can be visualized. The assay excludes completely the undesired fluctuation depending on the concentration of coexisting proteins and permits reliable and rapid quantitation of specific ER. Association constant and concentration of specific ER can be determined with 100 mg of mammary tumor tissue. The method has the additional advantage of not requiring expensive equipment.     

Separation of glycopeptides derived from brain glycoproteins by gel filtration.

Glycopeptides obtained from rat brain by proteolytic digestion with papain have been separated from glycosaminoglycans by means of gel filtration. The glycosaminoglycans appear in the void volume, whereas the glycopeptides are retarded. Glycopeptides of groups A+B (Brunngraber et al., 1973) (MW = 3800-500) C+D (MW = 2000) which were partially resolved by the method, were identified in the elution profile. Nucleic acids, also solubilized by papain, are eluted together with the glycosaminoglycans.