ChemInform Abstract: MEASUREMENT OF HYDROGEN EXCHANGE AT THE TRYPTOPHAN RESIDUES OF A PROTEIN BY STOPPED‐FLOW AND ULTRAVIOLET SPECTROSCOPY

Nakanishi, Mamoru; Nakamura, Hitomi; Hirakawa, Akiko Y.; Tsuboi, Masamichi; Nagamura, Toshihiko; Saijo, Yusuke

doi:10.1002/chin.197817089

Cited by 1 publication

(2 citation statements)

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“…Therefore, the difference intensity of fluorescence A F ( X ) is related to the ligand concentration SO according to (4) where f~ and fm denote the fluorescence intensity per molar concentration of free protein and protein-ligand complex, respectively. The binding constant K, can be determined from the intercept of the linear plot of l/So vs l/AF at 360 nm [ Fig.…”

Section: H-d Exchange Reaction For a Cross-linked Lysozyme Moleculementioning

confidence: 99%

“…On the other hand, Nakanishi et al 4 found that the H-D exchange reaction for the N-H group of tryptophan is measurable by the change in uv absorption at near 290 nm. Their method has also been used to follow the H-D exchange reaction for tryptophan residues in a folded lysozyme m~l e c u l e .~ Since the exchange rate for the N-H of a tryptophan residue exposed to the solvent is very fast, the exchange reaction for the residues buried in the interior of a protein molecule is governed by the unfolding rate of its region.…”

Section: Introductionmentioning

confidence: 98%

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Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

Segawa¹,

Nakayama²,

Sakane³

1981

Biopolymers

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SynopsisThe hydrogen-deuterium exchange reaction for the tryptophan residues in lysozyme have been followed in 4.5M LiBr at pH 7.2 in the temperature range of the unfolding transition by measuring the transmittance change at 293 nm. The exchange reaction proceeded in three phases at low temperature for native protein. The first and the second phases were ascribed to the H-D exchange reactions of three relatively exposed tryptophan residues on the molecular surface. The third phase corresponded to the H-D exchange reaction of the three tryptophan residues buried in the interior of the molecule. The H-D exchange reaction proceeded in two phases near the melting temperature and in a single phase at high temperature, where almost all molecules are unfolded. The H-D exchange of three tryptophan residues buried in folded molecules was caused by fluctuation between the folded and unfolded structure of the protein molecule. The rates of such a fluctuation were determined from the rates of the exchange reaction at various temperatures. These rates agreed very well with those determined from the temperature-jump method. This means that a protein molecule in solution fluctuates between the N-and D-states at every temperature within the transition region, where the N-form is the tightly folded native structure and the D-form the randomly coiled chain. From measurements of thermal unfolding of ester-108-lysozyme and the binding constant of (NAG)3 to ester-l08-lysozyme, it was found that almost all cross-linked molecules are in the folded state near 50°C and pH 7.2 in 4.5M LiBr, where intact molecules are unfolded. We also studied the H-D exchange reaction of ester-108-lysozyme. In the temperature region of 43-50°C, about 70% of the exchangeable tryptophan residues of ester-108-lysozyme were exchanged within 1 s immediately after the mixing of DzO, in spite of the fact that almost all molecules are in the folded state. This was considered the premelting of the surface of a cross-linked molecule.

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Section: H-d Exchange Reaction For a Cross-linked Lysozyme Moleculementioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

Segawa¹,

Nakayama²,

Sakane³

1981

Biopolymers

View full text Add to dashboard Cite

show abstract

ChemInform Abstract: MEASUREMENT OF HYDROGEN EXCHANGE AT THE TRYPTOPHAN RESIDUES OF A PROTEIN BY STOPPED‐FLOW AND ULTRAVIOLET SPECTROSCOPY

Cited by 1 publication

References 1 publication

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

Rates of structural fluctuations of lysozyme in the range of thermal unfolding transition

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