Triplet-Triplet Energy Transfer in α-Trypsin

ABSPIACT The fluorescence of the su posedly buried trtophan in ribonuclease T1 has been found to be collisionally quenched by acrylamide with a rate constant of 3 X 108 M-1 sec-1. Only a slight decrease in the quenching rate is observed upon a 5-fold increase in the viscosity of the solution. For this to be the case, the diffusion of the quencher must be limited by the protein matrix. To explain the process of diffusion through this complex material, the formation of "holes" in the lattice of a protein due to nanosecond fluctuations must be invoked. ThIus, the dynamic character of a protein molecule is revealed. The quenching rate constant has an activtion energy of 9 kcal/mol which can be used to charactedte the nature of the cohesive forces in the microenvironment about the indole ring. The mechanical properties of a portion of a protein matrix can, therefore, be described as one would for a fluid.Perhaps the most pressing problem facing the protein chemist today is to adequately describe the dynamic properties of proteins in solution, now that their static structure can be determined crystallographically. Evidence for some type of motion occurring at a time interval as small as a fraction of a nanosecond has been obtained from nuclear magnetic resonance (1), nitroxide spin labeling (2), dielectric relaxation (3), and various fluorescence studies (4-6). Depending on the time domain and experimental approach, descriptive terms such as "breathing" (7), "relaxation," "segmental motion," and "mobile defect" (8) have been advanced to portray the conformational "motility" (9) of proteins.Although it is recognized that fluctuations on the nanosecond time scale are allowed at the periphery of macromolecules, experimental evidence for such rapid motion of residues buried in "core" regions is not as available. In fact, the observation made by Longworth (10), that the lone tryptophan in ribonuclease T1 fluoresces with fine structure at room temperature, even suggests the absence of certain dynamic events in this protein. The enzyme was assayed by measuring the absorbance of trichloroacetic-acid-soluble material formed by enzymic digestion of the tRNA for 15 min at 30' at pH 7.5 (Tris buffer).Fluorescence-lifetime measurements were performed on an instrument resembling the model 9200 Nanosecond Fluorescence Spectrometer (ORTEC) constructed by S. Stevens and J. W. Longworth. Lifetimes were assigned by comparison of the raw decay data to simulated patterns that were synthesized by adding a theoretical single exponential decay curve to a measured system response. This was done using a computer program (also provided by S. Stevens and J. W. Longworth) that was designed to minimize the error between the raw and simulated curves in order to seek the best fit. The decays could be excellently described as single exponentials. The single component fits were at least as good as those obtained for pure samples of indole (4.3 nsec) and Nacetyltryptophanamide (2.8 nsec). During the 12-hr time period required to make the lifetime measu...

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“…A 500% decrease in the quenching rate was anticipated, but instead only a 20% drop in kq was observed § (see Fig. 1 (24).…”

Section: And Discussionmentioning

confidence: 99%

Dynamics of a protein matrix revealed by fluorescence quenching.

Eftink¹,

Ghiron²

1975

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

152

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“…Trypsin has four tryptophan residues (Trp51, Trp141, Trp215, and Trp237). Trp237 lies on the surface of the molecule and is thought not to contribute to the fluorescence of the enzyme 46. The presence of SDS alters the environment of the buried fluorophores, causing a slight red shift and decreasing their emission intensity.…”

Section: Resultsmentioning

confidence: 99%

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Prasad

Roy

2009

Biotechnology Progress

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Osmolytes have been reported to stabilize biomolecules and even whole organisms against exposure to adverse environmental conditions. In this work, we report for the first time the use of some of these osmolytes, viz., the disaccharides trehalose and sucrose, in the stabilization of bovine trypsin against exposure to the anionic detergent sodium dodecyl sulfate and autolysis. Exposure of trypsin to SDS at a molar ratio of 1:45 led to decrease in trypsin activity by 61%. In the presence of 1 M sucrose and 1 M trehalose, the residual trypsin activity was found to increase to that of original enzyme activity. These two disaccharides were also found to slow down the rate of autolysis, resulting in residual activities of 80 and 88%, respectively, after incubation for 24 h. Active site titration showed retention of the fraction of active sites in the presence of trehalose. Fluorescence and CD spectroscopies were used to decipher the probable mechanism of this protective role of the disaccharides. Although complete resumption of secondary structure was not seen in the presence of the two disaccharides, the spectra of trypsin in the presence of stabilizers resembled the spectrum of native trypsin and were significantly different from the spectrum of detergent-denatured enzyme. Correlating the data obtained from spectroscopy with those obtained from activity assay, we propose that the retention of secondary structure of the enzyme is largely responsible for the retention of the functionally active form of trypsin.

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Kinetics of triplet–triplet energy transfer and intramolecular distances in enzyme–inhibitor complexes

Galley¹,

Strambini²

1976

Nature

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Triplet-Triplet Energy Transfer in α-Trypsin

Cited by 7 publications

References 14 publications

Dynamics of a protein matrix revealed by fluorescence quenching.

Dynamics of a protein matrix revealed by fluorescence quenching.

Effect of disaccharides on the stabilization of bovine trypsin against detergent and autolysis

Kinetics of triplet–triplet energy transfer and intramolecular distances in enzyme–inhibitor complexes

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