Hydrogen-deuterium exchange of poly-dl-alanine in aqueous solution

Bryan, William P.; Nielsen, Sigurd O.

doi:10.1016/0006-3002(60)90843-x

Cited by 60 publications

(32 citation statements)

References 6 publications

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“…Measurements of the broadening of resonance lines by NMR techniques have revealed that the labile hydrogens of carboxyl, amino, guanidino, sulfhydryl, and hydroxyl groups exchange very rapidly in aqueous solution [see (151) for references], and that these exchange reactions are catalyzed by hydroxyl and hydrogen ions. Peptide hydrogen atoms, on the other hand, exchange usually at least 104 times more slowly than other groups (151,(156)(157)(158)(159). It is ger.erally agreed that the exchange of peptide hydrogens is catalyzed by hydroxyl and hydrogen ions (156)(157)(158)(159)(160), but recently Klotz Frank (159,160) have proposed that general acid-base catalysis occurs well.…”

Section: Hydrogen Exchangementioning

confidence: 96%

“…Peptide hydrogen atoms, on the other hand, exchange usually at least 104 times more slowly than other groups (151,(156)(157)(158)(159). It is ger.erally agreed that the exchange of peptide hydrogens is catalyzed by hydroxyl and hydrogen ions (156)(157)(158)(159)(160), but recently Klotz Frank (159,160) have proposed that general acid-base catalysis occurs well. In particular, thes,.~ investigators have found that the deuterium-hydrogen exchange of N-methyl acetamide in dioxane-deuterium oxide mixed solvent systems is catalyzed strongly by imidazole, tris (hydroxymethylamlnomethane), and by sev,~.ral carboxyllc acids.…”

Section: Hydrogen Exchangementioning

confidence: 96%

See 1 more Smart Citation

Physical Chemical Studies on Proteins and Polypeptides

Harrington¹,

Josephs²,

Segal³

1966

Annu. Rev. Biochem.

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Section: Hydrogen Exchangementioning

confidence: 96%

Section: Hydrogen Exchangementioning

confidence: 96%

Physical Chemical Studies on Proteins and Polypeptides

Harrington¹,

Josephs²,

Segal³

1966

Annu. Rev. Biochem.

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“…The 119 peptide protons are intrinsically slower. Infrared studies of Bryan and Nielsen (1960) on the hydrogen-deuterium exchange of poly-D,Lalanine show all the peptide protons to exchange in a single first-order reaction, which, together with other considerations, led these authors to suggest a random-chain conformation for this polypeptide in aqueous solution. (See also Elliott, 1962; but also Rosenheck and Doty, 1961).…”

Section: Studies With Ribonucleasementioning

confidence: 99%

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Sw¹

1963

Biochemistry

190

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A new method for measuring the hydrogen exchange of macromolecules in solution is described. The method uses tritium to trace the movement of hydrogen, and utilizes Sephadex columns to effect, in about 2 minutes, a separation between tritiated macromolecule and tritiated solvent great enough to allow the measurement of bound tritium. High sensitivity and freedom from artifact is demonstrated and the possible value of the technique for investigation of other kinds of colloidsmall molecule interaction is indicated. Competition experiments involving tritium, hydrogen, and deuterium indicate the absence of any equilibrium isotope effect in the ribonuclease-hydrogen isotope system, though a secondary kinetic isotope effect is apparent when ribonuclease is largely deuterated. Ribonuclease shows four clearly distinguishable kinetic classes of exchangeable hydrogens. Evidence is marshaled to suggest the independently measurable classes II, III, and IV (in order of decreasing rate of exchange) to represent "random-chain" peptides, peptides involved in α-helix, and otherwise shielded side-chain and peptide hydrogens, respectively.Protons attached to nitrogen, oxygen, and sulfur of molecules dispersed in liquid solution are, in general, able to exchange with mobile protons in the solvent. The rate of exchange of protons occupying specific molecular sites may be modified, or even largely controlled, by the details of molecular architecture in their immediate vicinity. Thus measurement of the kinetics of the hydrogen exchange of macromolecules can provide a fine probe for identifying and quantitating details of conformation and of changes in conformation of macromolecules.A practicable method for the measurement of hydrogen-exchange kinetics was first developed and used in the study of structure in proteins and polypeptides by Linderstrøm-Lang and his co-workers . Typically, in this method, deuterated protein is separated from deuterated solvent by freeze-drying, the protein is redissolved in water, and the kinetics of the loss of deuterium by the protein is followed by analyzing samples of solvent, after various times of protein incubation, for deuterium content. Deuterium content is determined by density measurement of tiny samples of solvent isolated from the protein by a second freeze-drying step. The method, though ingenious in conception, seems unusually demanding in execution and makes use of procedures which might be considered questionable. While the method is capable of considerable internal precision, a number of the operations involved, freezing, drying, heating (60°), redissolution,

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“…The rate of exchange, ko, of solvent exposed peptide groups, has been estimated from the exchange rate of the random coil peptide, poly-D,L-alanine (6,12,17). The exchange is catalyzed by protons, hydroxyl ions and water, and has a minimum rate about pH 3.0.…”

Section: Discussionmentioning

confidence: 99%

“…3.4 where the exchange rate has its minimum for most proteins (6,12,13). unusually broad amide II band was determined by the difference spectra indicated in the insert in Figure 1, and it has a maximum about 1540…”

Section: Methodsmentioning

confidence: 99%

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

Dreyer

Ottesen

Hvidt

1986

Carlsberg Res. Commun.

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Keywords: Yeast Cu2,Zn2-superoxide dismutase, apo-superoxide dismutase, hydrogendeuterium exchange, metal-dependent structural transition, E X 2 -m e c h a n i s mThe role of copper and zinc for the structural properties of yeast Cu2,Zn2-SOD was studied by measuring the hydrogen-deuterium exchange in peptide groups of Cu:,Zn:-SOD, Cu:,E2-SOD, E:,Zn2-SOD and apo-SOD. Apo-SOD displays remarkably rapid exchange kinetics as compared to Cu2,Zn2-SOD, indicating a less stable structural organization after metal removal. At neutral and alkaline pD the exchange rates are in agreement with the EX2 mechanism of exchange; the exchange rates at low pD suggest a structural transition. In Cu2,Zn2-SOD this transition occurs at pD about 4.5, and in apo-SOD at pD about 6.2, 1.7 pD units higher. Binding of either copper or zinc to apo-SOD results in reductions of the exchange rate, which is further reduced after full reconstitufion to Cu_~,Zn.,-SOD, demonstrating that copper and zinc are equally important for the structural stability of the protein molecule. I N T R O D U C T I O NCu_,,Znz-SOD from yeast is a member of a highly conserved group of enzymes found in the cytosol of eucaryotic cells, believed to act as scavengers of toxic superoxide ions, O~, formed during oxidative metabolism. Yeast Cu2,Zn2-SOD has a molecular weight of 31,900, and it is composed of two identical subunits each containing a bimetallic Cu-Zn-center, which is essential for the catalytic mechanism (8,15,22). The X-ray crystal structure of bovine erythrocyte Cu2,Zn2-SOD at 2A resolution shows that each subunit consists of an eight stranded [3-bartel of Greek key topology, suggesting a very rigid structure. The copper is liganded to four histidines in a distorted square plane, and the zinc is liganded to three histidines and an aspartic acid in a tetrahedral geometry (28). 'H-NMR studies indicate close similarity in the metal binding sites of bovine erythrocyte and yeast Cu2,Zn2-SOD (8), although a metal bridging histidine in the bovine enzyme is apparently not liganded to zinc in the yeast enzyme (3).It is now well established that copper is essential for the catalysis of superoxide dismutation, during which copper is alternately oxidized and Abbreviations: Cu_,,E2-SOD = di-copper superoxide dismutase; E2,Zn2-SOD = di-zinc superoxide dismutase; SOD = superoxide dismutase.Springer-Verlag

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Hydrogen-deuterium exchange of poly-dl-alanine in aqueous solution

Cited by 60 publications

References 6 publications

Physical Chemical Studies on Proteins and Polypeptides

Physical Chemical Studies on Proteins and Polypeptides

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

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Hydrogen-deuterium exchange of poly-dl-alanine in aqueous solution

Cited by 60 publications

References 6 publications

Physical Chemical Studies on Proteins and Polypeptides

Physical Chemical Studies on Proteins and Polypeptides

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease*

Hydrogen-deuterium exchange in yeast Cu2,Zn2-superoxide dismutase and apo-superoxide dismutase

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Product

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About

A Hydrogen Exchange Method Using Tritium and Sephadex: Its Application to Ribonuclease^*