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C A Ghiron

Publications and source records attributed to C A Ghiron.

32 records · Page 2Linked to original sources
PubMed1978

Flash photolysis of human serum albumin: characterization of the indole triplet absorption spectrum and decay at ambient temperature.

The method of flash photolysis was used to identify the transient absorption spectrum and to characterize the decay kinetics of the indole triplet of human serum albumin. This protein was studied because it contains a single indole side chain which is deeply buried in an expandable oily region and because the phosphorescence of the homologous indole in bovine serum albumin could not be detected at ambient temperatures. The transient was identified on the following basis: (i) its triplet-triplet absorption spectrum was similar to those previously reported for indole and tryptophan; (ii) it was quenched by small quantities of oxygen; and (iii) it was photobleached by 370- to 700-nm light. In a nitrogen-saturated solution at room temperature, the indole triplet decays exponentially for more than a factor of 10 with a lifetime of 0.5 msec. These observations suggest that, because of its exponential decay and relatively long lifetime, the triplet will be more valuable than the indole singlet as an intrinsic reporter group for the study of the structure and dynamics of proteins in solution.

Humans↗
PubMed1977

Exposure of tryptophanyl residues and protein dynamics.

The acrylamide quenching reaction is shown to be very discriminating in sensing the exposure of fluorescing tryptophanyl residues in globular proteins. The quenching rate constants for some proteins, such as aldolase and human serum albumin, are reported to be independent of the solvent viscosity, indicating that the reaction is limited by penetration of the quencher through the protein matrix. Temperature-dependent studies are performed to determine the activation energy and entropy for the penetration of acrylamide into these proteins. The tryptophanyl residues in aldolase are shown to be shielded by a large activation energy barrier, while the single residue in human serum albumin is shielded by a large activation entropy barrier. These parameters characterize the nature of the protein matrix enveloping the fluorophors.

Acrylamides↗
PubMed1977

A hydrophobic quencher of protein fluorescence: 2,2,2-trichloroethanol.

Previously the neutral fluorescence quenching probe, acrylamide, was employed to determine the degree of exposure of tryptophan residues in proteins. A less polar neutral quencher 2,2,2-trichloroethanol (trichloroethanol) was used in the present work to investigate whether it would preferentially interact with apolar regions of proteins. For most proteins studied, the degree of quenching by trichloroethanol is found to be about the same as with acrylamide. However, for human and bovine serum albumin hydrophobic interactions between trichloroethanol and these proteins occur, leading to an exalted quenching. The fluorescence quencher thus senses the presence of a hydrophobic domain in the vicinity of the tryptophan residues in these proteins. Trichloroethanol is shown to induce conformational changes in certain proteins and to be a potentially useful quencher for proteins having predominantly tyrosine emission.

Acrylamides↗
PubMed1976

Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies.

Acrylamide is an efficient quencher of tryptophanyl fluorescence which we report to be very discriminating in sensing the degree of exposure of this residue in proteins. The quenching reaction involves physical contact between the quencher and an excited indole ring, and can be kinetically described in terms of a collisional and a static component. The rate constant for the collisional component is a kinetic measure of the exposure of a residue in a protein, and values ranging from 4 X 10(9) M-1 S-1 for the fully exposed tryptophan in the polypeptide, adrenocorticotropin, to less than 5 X 10(8) M-1 S-1 for the buried residue in azurin have been found. Static quenching is readily detected in proteins that are denatured, or contain only a single fluorophor. Quenching patterns for most multi-tryptophan containing proteins are difficult to analyze precisely, but qualitative information can, nevertheless, be extracted. Applications of this probing technique for monitoring protein conformational changes, such as the acid-induced expansion of human serum albumin, and inhibitor binding to enzymes, are presented. The value of this method lies in its ability to sense not only the steady-state exposure of a residue in a protein, but also its dynamic exposure.

Acrylamides↗
PubMed1975

Dynamics of a protein matrix revealed by fluorescence quenching.

The fluorescence of the supposedly buried tryptophan in ribonuclease T1 has been found to be collisionally quenched by acrylamide with a rate constant of 3 X 10(8) 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. Thus, the dynamic character of a protein molecule is revealed. The quenching rate constant has an activation energy of 9 kcal/mol which can be used to characterize 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.

Acrylamides↗
PubMed1973

Triplet-triplet energy transfer in alpha-trypsin.

Experiments are reported that demonstrate that light absorbed by ionized tyrosinyl sensitizes the phosphorescence of tryptophanyl residues of native alpha-trypsin. The sensitization effect is abolished when alpha-trypsin is unfolded in guanidine hydrochloride. Under the experimental conditions used, the tryptophan phosphorescence could only have been induced by an electron-exchange interaction. These results, therefore, require that there be at least one ionized tyrosinyl-tryptophanyl pair in the native enzyme and that the distance between the two side chains be sufficiently short to permit electron exchange.

Amidines↗