J. B. Alexander Ross

Time-resolved fluorescence of the two tryptophans in horse liver alcohol dehydrogenase

The tryptophan fluorescence decay of horse liver alcohol dehydrogenase, at 10 degrees C in 0.1 M pH 7.4 sodium phosphate buffer, with excitation at 295 nm, is a double exponential with time constants of 3.8 and 7.2 ns. Within experimental error, the two lifetimes remain constant across the emission spectrum. Only the 3.8-ns lifetime is quenched in the NAD+-pyrazole ternary complex, and only the 7.2-ns lifetime is quenched by 0-0.05 M KI. On the basis of these results, we assign the 3.8-ns lifetime to the buried tryptophan, Trp-314, and the 7.2-ns lifetime to the exposed tryptophan, Trp-15. The steady-state lifetime-resolved emission spectrum of Trp-15 has a maximum at approximately 340 nm and that of Trp-15 is at approximately 325 nm. The total time-resolved emission, after 40 ns of decay, has a maximum between 338 and 340 nm and is primarily due to the Trp-15 emission. As a consequence of the wavelength dependence of the preexponential weighting factors, there is an increase in the average lifetime from the blue to the red edge of the emission. This increase reflects the change in the spectral contributions of Trp-15 and Trp-314. Consideration of the spectral overlap between the emission spectra of the two tryptophans and the absorption due to formation of the ternary complex, as well as the distances between the two residues and the bound NAD+, shows that the selective fluorescence quenching in the ternary complex can be accounted for entirely by singlet-single energy transfer. The decay of the fluorescence anisotropy was measured as a function of temperature from 10 to 40 degrees C and is well described by a monoexponential decay law. Over this temperature range the calculated hydrodynamic radius increases from 33.5 to 35.1 A. Evidently, the indole groups of Trp-15 and Trp-314 rotate with the protein as a whole, and there is some expansion of the protein matrix as the ambient temperature is increased.

Current status of the molecular structure and function of the plasma sex steroid-binding protein (SBP)☆

Purification and characterization of the sex steroid-binding protein (SBP) from human, macaque, baboon, and rabbit sera indicate that the protein is composed of two polypeptide chains which associate noncovalently to yield a native structure having molecular weight distributions of about 88,000 for primate SBPs, and 80,000 for rabbit SBP. The subunit molecular weight distributions are 44,000 for human SBP, 47,000 for macaque and baboon SBPs, and 40,000 for rabbit SBP. Isoelectric focusing show extensive microheterogeneity for all four SBPs. The patterns appear to be unique for each species and reveal the presence of at least twelve bands of different colour intensity reflecting a specific spectrum of active SBP molecules. The existence of the large number of dimeric forms of SBP arises through the combination of many variants of the same two subunits containing different amounts and types of carbohydrate sidechains. Physiological studies on the intravenous infusion of pure rhesus SBP, human SBP, and purified monospecific SBP-antibodies into the rhesus reveal an inverse relationship between SBP and the metabolic clearance rate of testosterone. The effect is complex and depends on the concentration of SBP, albumin, and testosterone which in turn influences the distribution of testosterone between albumin and SBP.

Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides

The fluorescence and phosphorescence of proteins and polypeptides is the sum of the contributions from the three aromatic amino acids tryptophan, tyrosine, and phenylalanine. The work on protein and polypeptide luminescence prior to 1971 has been reviewed in detail by Longworth. Another fine account of the early work, emphasizing tryptophan and tyrosine, is the monograph by Konev. An excellent review on tyrosine fluorescence in proteins and model peptides, for the period up to 1975, is given by Cowgill. In 1984, Creed reviewed the photophysics and photochemistry of tyrosine and its simple derivatives, including a thorough coverage of steady-state fluorescence and a brief discussion of triplet-state properties, but did not include any work on proteins or polypeptides. The first quantitative studies of the excited-state properties of the three aromatic amino acids were carried out in the 1950s. The low-temperature phosphorescence of the aromatic amino acids was initially observed by Debye and Edwards in 1952, and phosphorescence emission spectra were reported by Steele and Szent-Gyorgyi in 1957. In 1953, Weber postulated that the fluorescence of the aromatic amino acids should occur in the near-ultraviolet region of the electromagnetic spectrum. In 1956, independently and almost simultaneously, Duggan and Udenfriend and Shore and Pardee reported the results of their investigations of protein fluorescence. At the same time,

Molecular characterization of the sex steroid binding protein (SBP) of plasma. Re-examination of rabbit SBP and comparison with the human, macaque and baboon proteins.

Physico-chemical characterization of the sex steroid-binding protein, SBP, of rabbit plasma reveals that it is a dimer of mol. wt 85,800 composed of similar subunits of mol. wt 43,000. These data confirm our original proposal for a dimeric structure. The protein contains 9% carbohydrate, comprised of mannose, galactose, N-acetylglucosamine and sialic acid. It is devoid of N-acetylgalactosamine and fucose. The protein binds one molecule of 5 alpha-dihydrotestosterone per dimer with a Kd of 0.89 nM (12 degrees C). Comparison with the human, monkey and baboon SBPs indicates that all these proteins have the same dimeric molecular organization and exhibit microheterogeneity in SDS-PAGE and isoelectricfocusing. Rabbit SBP, however, contains less carbohydrate and has a higher polypeptide molecular weight than all the other SBPs. Spectrophotometric data also indicate that some tryptophan residues are in a different chemical environment than those in other SBPs. The observed microheterogeneity in all four SBP species is due for the most part to variable glycosylation of the subunit and variability at the amino-terminal region of the subunit. Combination of these and other phenomena will generate a significant number of isomeric forms of the SBP subunit which will then interact stoichiometrically to yield active dimeric SBP molecules. These differ slightly from each other depending upon the charge and size of the subunit comprising the dimeric structure, and will result in the observed microheterogeneity of pure SBP preparations. Based on these results along with more recent amino acid sequence data, we conclude that all four SBPs are dimers composed of identical polypeptide chains.

Dynamics of Biomolecules: Assignment of Local Motions by Fluorescence Anisotropy Decay

Many biological systems have multiple fluorophores that experience multiple depolarizing motions, requiring multiple lifetimes and correlation times to define the fluorescence intensity and anisotropy decays, respectively. To simplify analyses, an assumption often made is that all fluorophores experience all depolarizing motions. However, this assumption usually is invalid, because each lifetime is not necessarily associated with each correlation time. To help establish the correct associations and recover accurate kinetic parameters, a general kinetic scheme that can examine all possible associations is presented. Using synthetic data sets, the ability of the scheme to discriminate among all nine association models possible for two lifetimes and two correlation times has been evaluated. Correct determination of the association model, and accurate recovery of the decay parameters, required the global analysis of related data sets. This general kinetic scheme was then used for global analyses of liver alcohol dehydrogenase anisotropy data sets. The results indicate that only one of the two tryptophan residues in each subunit is depolarized by process(es) independent of the enzymes rotations. By applying the proper kinetic scheme and appropriate analysis procedures to time-resolved fluorescence anisotropy data, it is therefore possible to examine the dynamics of specific portions of a macromolecule in solution.

Wavelength dependence of the zero-field splittings in the triplet state of tryptophan☆

Abstract It is shown that abrupt discontinuities occur in the zero-field splitting parameters of the isolated amino acid, tryptophan, and its derivatives as a function of the wavelength of the optically detected emission from the excited triplet state. These discontinuities are attributed to the convolution of the magnetic transitions associated with each of the elementary vibronic components assuming that the frequency of the magnetic transition is a linear function of the wavelength for each elementary vibronic component.

LINKED-FUNCTION ANALYSIS OF FLUORESCENCE DECAY KINETICS: RESOLUTION OF SIDE-CHAIN ROTAMER POPULATIONS OF A SINGLE AROMATIC AMINO ACID IN SMALL POLYPEPTIDES

A linked‐function approach to fluorescence decay data analysis is presented that permits complex systems to be resolved from a single decay curve. The method involves linking fluorescence decay parameters based on a relationship established by independent physical measurements. As an example, by correlating the fluorescence data with 1H‐NMR results, the complex fluorescence decay kinetics of tyrosine analogs and single tyrosyl residues in simple polypeptides can be explained by ground‐state rotameric populations of the phenol ring about the Cα‐Cβ bond.

Estradiol entry into endometrial cells in suspension.

Cells from a human endometrial adenocarcinoma cell line (HEC-50) were superfused with mixtures of [3H]E2 and [14C]E1 in order to estimate rates of entry and exit of E1 and E2 into and out of cells according to previously published procedures (J. steroid Biochem., 13 (1980) 1379). Proportionality between rates of entry and concentrations of E2 outside the cells, indicative of passive diffusion, was found at levels of E2 ranging from 1 to 100 ng/ml. Effects of albumin and of pure human sex steroid binding protein (SBP) on the rate of entry of E2 were also evaluated in parallel superfusions. In other single tracer experiments, [3H]E2 was used at concentrations as low as 100 pg/ml and the effects of plasma proteins on entry were evaluated by measuring steady-state concentrations of E2 and E1 in cells and superfusate. Results from these experiments indicate that albumin, and to a larger extent SBP, reduced the entry of E2 into HEC-50 cells. Similar results were obtained when CG-5 cells, a variant of the human breast cancer cell line MCF-7, were superfused with [3H]E2. Further experiments are needed, however, to determine the physiologic role of plasma estrogen binding proteins on the entry and metabolism of E1 and E2 into target cells.

[12-Homoarginine]glucagon: synthesis and observations on conformation, biological activity, and copper-mediated peptide cleavage.

Specific modification of the single lysine residue (Lys-12) in glucagon with O-methylisourea has been effected by blocking the reactivity of the amino terminal histidine with copper, providing a method for obtaining [12-homoarginine]glucagon. It was found that as a side reaction, under the conditions of the modification reaction, Cu(II) catalyzed cleavage of the polypeptide chain between Asp-9 and Tyr-10, and between Lys-12 and Tyr-13. This observation may be of value for development of a sequence-specific peptide cleavage procedure. The dilute solution conformations of glucagon and [12-homoarginine]-glucagon were compared by circular dichroism, fluorescence, phosphorescence, energy transfer, and optical detection of magnetic resonance. The results indicate that conversion of Lys-12 to homoarginine does not alter the helix content the side chain conformation in the vicinity of the tyrosine and tryptophan residues, or the relative distances and orientations between these residues. However, the modification reduces the hormone potency towards activation of lipolysis in isolated rat epididymal fat cells by a factor of seven. We attribute the loss of potency to an interference with a specific interaction between the lysine residue and the fat cell hormone receptor, and not to a change in the solution conformation of the hormone.

Cytochrome c Can Form a Well-Defined Binding Pocket for Hydrocarbons.

Cytochrome c can acquire peroxidase activity when it binds to cardiolipin in mitochondrial membranes. The resulting oxygenation of cardiolipin by cytochrome c provides an early signal for the onset of apoptosis. The structure of this enzyme-substrate complex is a matter of considerable debate. We present three structures at 1.7-2.0 Å resolution of a domain-swapped dimer of yeast iso-1-cytochrome c with the detergents, CYMAL-5, CYMAL-6, and ω-undecylenyl-β-d-maltopyranoside, bound in a channel that places the hydrocarbon moieties of these detergents next to the heme. The heme is poised for peroxidase activity with water bound in place of Met80, which serves as the axial heme ligand when cytochrome c functions as an electron carrier. The hydroxyl group of Tyr67 sits 3.6-4.0 Å from the nearest carbon of the detergents, positioned to act as a relay in radical abstraction during peroxidase activity. Docking studies with linoleic acid, the most common fatty acid component of cardiolipin, show that C11 of linoleic acid can sit adjacent to Tyr67 and the heme, consistent with the oxygenation pattern observed in lipidomics studies. The well-defined hydrocarbon binding pocket provides atomic resolution evidence for the extended lipid anchorage model for cytochrome c/cardiolipin binding. Dimer dissociation/association kinetics for yeast versus equine cytochrome c indicate that formation of mammalian cytochrome c dimers in vivo would require catalysis. However, the dimer structure shows that only a modest deformation of monomeric cytochrome c would suffice to form the hydrocarbon binding site occupied by these detergents.