Rapid microdialysis and hydrogen exchange

Englander, S. W.; Crowe, D. M.

doi:10.1016/0003-2697(65)90225-3

Cited by 98 publications

(30 citation statements)

References 2 publications

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“…Immediately after the isotopic content of the medium is changed, the mole fractions of the bound hydrogen isotopes begin readjusting to the isotopic distribution of the new medium, and the rate at which equilibrium is approached depends upon the secondary and tertiary structure of the exchanging protein. Rapid changes of the medium have been effected by adding tracer amounts of tritium to the protein solution (163), by dissolving a lyophilized sample in solvent with isotopic content which differs from that of the sample (164,165), by passing a sample which is in equilibrium with tritium through Sephadex G-25 column (154), and by dialysis (166). At appropriate intervals aliquots of exchanging sample are assayed for isotopic content by density measurements (165), by liquid scintillation counting (154,163,166), or infrared spectrometry (161,164,167).…”

Section: Methods Employed In Exchangementioning

confidence: 99%

Physical Chemical Studies on Proteins and Polypeptides

Harrington¹,

Josephs²,

Segal³

1966

Annu. Rev. Biochem.

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Section: Methods Employed In Exchangementioning

confidence: 99%

Physical Chemical Studies on Proteins and Polypeptides

Harrington¹,

Josephs²,

Segal³

1966

Annu. Rev. Biochem.

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“…The factor T depends on the crystal shape and on the desired degree of diffusional equilibrium, i.e. the permissible fraction of residual tritiated water, ~ For Mo M___L less than 0.1, T may be calculated from Mo equation [4], which is derived from expressions given by CRANK (2):…”

Section: Initial Rinsing Proceduresmentioning

confidence: 99%

“…Unfortunately, the advantage of direct measurement of the hydrogen replacement in specific categories of exchanging sites by the spectroscopic methods seems unattainable in suspensions of insoluble protein due to demands for homogeneity or freedom for molecules to rotate. The tritium tracer technique is much more easily adapted to such proteins, since the only basic requirement is a convenient method for separating the protein from label contained in the solvent, i. e. cryosublimation (10, l l), gel filtration (5), dialysis (4) or adsorption of the protein to phosphocellulose paper (17).…”

Section: Introductionmentioning

confidence: 99%

A simple hydrogen exchange method for cross-linked protein crystals

Tüchsen

Ottesen

1979

Carlsberg Res. Commun.

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A method is presented for measurement of hydrogen-tritium exchange in insoluble protein samples using a specially constructed closed chamber type filtration device. The protein sample is initially exhaustively tritiated at high pH, and back-exchange is initiated in the filtration unit by removal of tritiated solvent from the sample by rinsing with buffer of desired pH. The back-exchange is followed by sequential samplings of the tritium liberated to the solvent and it is completed by increasing pH. Experiments with monoclinic hen egg white lysozyme crystals, insolubilized by cross-linking with glutaraldehyde, could be followed as early as 20 seconds from initiation of back-exchange and exchange curves were highly reproducible. Exchange curves were identical with those of the dissolved enzyme at pH 3 and only small differences were detected between the two states at pH 6.8, supporting the idea of negligible change of protein motility upon crystallization and cross-linking. Diffusional equilibration processes are discussed.

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“…Reactions were terminated by cooling on ice for 5 min and precipitating with 2 vol of cold ethanol. Free 3H20 and fastexchanging tritium were removed by rapid dialysis (9). The [3H]tRNAPhe was isolated by phenol extraction and sucrose gradient centrifugation and was purified by preparative gel electrophoresis (10).…”

Section: Methodsmentioning

confidence: 99%

Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange.

Farber¹,

Cantor²

1980

Proc. Natl. Acad. Sci. U.S.A.

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The rate of incorporation of tritium from the solvent into the C-8 position of purines in RNA is markedly sensitive to the microenvironment. This slow tritium exchange reaction has been used to study the structure and interactions of yeast tRNAPhe bound to poly(U)programed tight-couple 70S ribosomes of Escherichia coli. The tritium incorporation into specific sites 6f the tRNA was determined by enzymatic digestion and measurement of the specific activity of each of the isolated radioactive fragments. 1 for a review). Models using this structure have been proposed (2-4) to describe the molecular motions involved in the translocation of tRNA during protein synthesis. However, the ribosome-bound structure may not be the same as that of tRNA free in solution. The overall shape of tRNA is maintained by various tertiary interactions (5), and a number of studies indicate that small perturbations on the structure can disrupt this delicate network. The effect of ribosome binding on tRNA structure is unknown.We have approached these two problems by comparing the rates of tritium exchange into free and ribosome-bound tRNA. This nonperturbing method is based on the innocuous replacement of the C-8 proton of purine bases with tritium from the solvent (6). Free tRNA and tRNA-ribosome complexes were incubated in a 3H20-containing buffer at 37'C. Then, after the free 3H20 and rapidly exchanging tritium had been removed, the incorporation rates into individual sites were measured. MATERIALS AND METHODSYeast tRNAPhe (amino acid acceptance of 1452 pmol/A2w unit) and Escherichia coli tRNAGIU (1126 pmol/A2r0 unit) were obtained from Boehringer Mannheim. 70S tight-couple ribosomes from E. coli MRE600 were purified by zonal centrifugation (7) and reactivated (8) prior to use. To study ribosomebound tRNA, ribosomes were incubated with poly(U) (Enzo Biochem, New York) at 37°C for 15 min in CAC buffer (10 mM sodium cacodylate, pH 7.0/20 mM Mg(OAc)2/50 mM NH4Cl/6 mM 2-mercaptoethanol). A stoichiometric amount of yeast tRNAPhe was added and incubated for 20 min. 3H20 (12-15 Ci/ml, New England Nuclear; 1 Ci = 3.7 X 1010 becquerels) was added to yield a final specific activity of 2-2.5 Ci/ml. Typical final concentrations were 50 mg of ribosomes, 4 mg of poly(U), and 8 A260 units of tRNAPhe in 1 ml of CAC buffer. All buffers were filtered through a 0.45-/im-pore Millipore filter and the tRNA and poly(U) were extracted with phenol prior to use. The complex was incubated at 37°C for 27 hr. Reactions were terminated by cooling on ice for 5 min and precipitating with 2 vol of cold ethanol. Free 3H20 and fastexchanging tritium were removed by rapid dialysis (9). The [3H]tRNAPhe was isolated by phenol extraction and sucrose gradient centrifugation and was purified by preparative gel electrophoresis (10).Free tRNAPhe was typically incubated in 10 mM sodium cacodylate, pH 7.0/10 mM Mg(OAc)2/100 mM NaCl/1 mM EDTA containing 2.5-3.0 Ci of 3H20 per ml for 26 hr at a concentration of 10 mg/ml. In one reaction tRNAPhe was tritiated in the presence...

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Rapid microdialysis and hydrogen exchange

Cited by 98 publications

References 2 publications

Physical Chemical Studies on Proteins and Polypeptides

Physical Chemical Studies on Proteins and Polypeptides

A simple hydrogen exchange method for cross-linked protein crystals

Comparison of the structures of free and ribosome-bound tRNAPhe by using slow tritium exchange.

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