A simplified electrophoretic system for determining molecular weights of proteins.
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ABSTRACT:
SUBMITTER: Manwell C
PROVIDER: S-EPMC1164931 | biostudies-other | 1977 Sep
REPOSITORIES: biostudies-other
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Project description:1. Rats were injected with [U-(14)C]glucose and after various intervals extracts of whole brain proteins (and in some cases proteins from liver, blood and heart) were prepared by high-speed centrifugation of homogenates in 0.9% sodium chloride or 0.5% sodium deoxycholate. 2. The extracts were subjected to gel filtration on columns of Sephadex G-200 equilibrated with 0.9% sodium chloride or 0.5% sodium deoxycholate. 3. Extracts prepared with both solvents displayed on gel filtration a continuous range of proteins of approximate molecular weights ranging from less than 2x10(4) to more than 8x10(5). 4. The relative amount of the large proteins (mol.wt.>8x10(5)) was conspicuously higher in brain and liver than in blood. 5. At 15min after the injection of [U-(14)C]glucose the smaller protein molecules (mol.wt.<2x10(4)) were significantly radioactive, whereas no (14)C could be detected in the larger (mol.wt.>2x10(4)) protein molecules. The labelling of all protein samples was similar within 4h after injection of [U-(14)C]glucose. Fractionation of brain proteins into distinctly different groups by the methods used in the present work yielded protein samples with a specific radioactivity comparable with that of total brain protein. 6. No evidence could be obtained by the methods used in the present and previous work to indicate the presence of a significant amount of ;metabolically inert protein' in the brain. 7. It is concluded that: (a) most or all of the brain proteins are in a dynamic state of equilibrium between continuous catabolism and anabolism; (b) the continuous conversion of glucose into protein is an important part of the maintenance of this equilibrium and of the homoeostasis of brain proteins in vivo.
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Project description:Issues of molecular weight determination have been central to the development of supramolecular polymer chemistry. Whereas relationships between concentration and optical features are established for well-behaved absorptive and emissive species, for most supramolecular polymeric systems no simple correlation exists between optical performance and number-average molecular weight (M n). As such, the M n of supramolecular polymers have to be inferred from various measurements. Herein, we report an anion-responsive supramolecular polymer [M1·Zn(OTf)2]n that exhibits monotonic changes in the fluorescence color as a function of M n Based on theoretical estimates, the calculated average degree of polymerization (DPcal) increases from 16.9 to 84.5 as the monomer concentration increases from 0.08 mM to 2.00 mM. Meanwhile, the fluorescent colors of M1 + Zn(OTf)2 solutions were found to pass from green to yellow and to orange, corresponding to a red shift in the maximum emission band (λmax ). Therefore, a relationship between DPcal and λmax could be established. Additionally, the anion-responsive nature of the present system meant that the extent of supramolecular polymerization could be regulated by introducing anions, with the resulting change in M n being readily monitored via changes in the fluorescent emission features.
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Project description:1. Correlation between elution volume, V(e), and molecular weight was investigated for gel filtration of proteins of molecular weights ranging from 3500 (glucagon) to 820000 (alpha-crystallin) on Sephadex G-200 columns at pH7.5. 2. Allowing for uncertainties in the molecular weights, the results for most of the carbohydrate-free globular proteins fitted a smooth V(e)-log(mol.wt.) curve. In the lower part of the molecular-weight range the results were similar to those obtained with Sephadex G-75 and G-100 gels. 3. V(e)-log(mol.wt.) curves based on results with the three gels are taken to represent the behaviour of ;typical' globular proteins, and are proposed as standard data for the uniform interpretation of gel-filtration experiments. 4. Some glycoproteins, including gamma-globulins and fibrinogen, do not conform to the standard relationship. The effect of shape and carbohydrate content on the gel-filtration behaviour of proteins is discussed. 5. As predicted by the theoretical studies of other authors, correlation exists between the gel-filtration behaviour and diffusion coefficients of proteins. 6. The lower molecular-weight limit for complete exclusion of typical globular proteins from Sephadex G-200 varies with the swelling of the gel, but is usually >10(6). 7. The concentration-dependent dissociation of glutamate dehydrogenase was observed in experiments with Sephadex G-200, and the sub-unit molecular weight estimated as 250000. The free sub-units readily lose enzymic activity. 8. Recognition of the atypical gel-filtration behaviour of gamma-globulins necessitates an alteration to several molecular weights previously estimated with Sephadex G-100 (Andrews, 1964). New values are: yeast glucose 6-phosphate dehydrogenase, 128000; bovine intestinal alkaline phosphatase, 130000; Aerobacter aerogenes glycerol dehydrogenase, 140000; milk alkaline phosphatase, 180000.
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