Gregorio Weber

Rotational Brownian Motion and Polarization of the Fluorescence of Solutions

Publisher Summary The chapter discusses the rotational Brownian motion and polarization of the fluorescence of solutions. It is possible to learn something of the size and shape of protein molecules by studying their Brownian motion. Such motion involves the translation of the molecules but also their rotational movements. The Brownian motion is not observed from a study of the physical properties of the solution. When the dissolved molecules are fluorescent, a study of the polarization of the emitted radiation gives important information about rotational motion. At any instant, all possible directions are equally represented, and the molecules are thus randomly disposed. In fact, unity at the initial instant is where there is perfect orientation and reaches zero value when the orientation is random.

Energetics of Ligand Binding to Proteins

Publisher Summary The interaction of proteins with small ligands embodies the fundamental physicochemical principles that are operative in the physiological regulation of enzyme activity, the specific interactions among proteins and of proteins with nucleic acids and other macromolecules. This chapter describes the multiple ligand binding by proteins, binding by multimer proteins, extension of the concept of ligand interaction to covalent bond exchange, cooperativity and ligand correlation, and biological specificity and ligand binding. Many parts of the compact protein structure can contribute toward the shift in energy and structure taking place on ligand binding. The energies of ligand–ligand interaction, although small are sufficient to shift the covalent equilibria in which proteins take part, to a significant extent, and this may be found responsible for the interconversion of chemical and osmotic energies in metabolism. While, the ligand correlation because of the existence of free-energy coupling among the bound ligands is a molecular property to be explained by a study of the isolated protein, the enhancement of the effects by the simultaneous changes in ligand concentration is a system property that requires consideration of other entities. The consideration of ligand–protein interactions provides with a model for the relative importance to be assigned to molecules in the organic functions. Thus, study of the interactions of proteins and small ligands provides the basis for the understanding of biological specificity at the molecular level.

The effect of high pressure upon proteins and other biomolecules

We shall not attempt here to enumerate the results or review in a systematic way the significant literature dealing with the use of high pressure in studies of proteins and other molecules of biological interest. Two recent reviews on this subject, one by MOrild (1981) and another by Heremans (1982), and a further article by Jaenicke (1981) on enzymes under extreme environmental conditions contain expositions and references that would render redundatn such a task. Rather we concentrate here on the examination of othe conceptual framework employed in the interpretation of high pressure experiments and in the critical discussion of our knowledge of selected areas of present interest and likely future significance.

Total Quantum Flux of Isotropic Sources

The total radiant flux from a solution containing a radioactive compound, hexadecane-1-C14, plus an appropriate scintillator has been determined. The procedure used involves comparison of its luminescent emission with the light scattered by glycogen illuminated with a monochromatic homogeneous light beam of known photon flux. From the result obtained the scintillation quantum yield of β− disintegration from carbon 14 has been determined to be 793 photons; thus 5.25% of the energy appears as light.

RESOLUTION OF THE FLUORESCENCE EXCITATION SPECTRUM OF INDOLE INTO THE 1La AND 1Lb EXCITATION BANDS

Abstract— The fluorescence excitation spectrum and the excitation polarization spectrum of indole in propylene glycol were measured at — 58°C, after selecting by optical filters the emission originating from the 1La electronic level. From the analysis of these spectra, the excitation spectrum was resolved into the 1La and 1La excitation bands. A similar resolution of the excitation spectrum of tryptophan is given. This method can also be applied to the resolution of the emission spectrum in cases of dual emission.

Influence of Brownian Rotations and Energy Transfer upon the Measurements of Fluorescence Lifetime

The depolarization of the fluorescence of solutions by either Brownian rotations or intermolecular energy transfer may be simply described by a system of first‐order linear differential equations containing as only parameters the rate of fluorescence emission and the rate of transport of the excitation from one orthogonal component of the emission to another. The steady‐state solution has the form of Perrins equation describing the depolarization by Brownian rotations, and the time‐dependent depolarization following a unit light impulse is that originally described by Jablonski. The solution for sinusoidal excitation is novel in that: 1. It shows the difference in lifetime between the polarized components of the emission to be a sensitive function of the ratio of the modulation frequency ω to the emission rate λ. For ω/λ > 1 the difference between the polarized lifetimes may become many times greater than that observed after a unit light impulse. 2. It permits the determination of both the rate of transp...

Estimation of the polarity of the protein interior by optical spectroscopy

Crystallographic studies of myoglobin have shown that the haem pocket is lined with nonpolar amino-acid residues1. In order to estimate the true polarity of the interior of proteins, certain studies have used bound fluorophores2–7, the spectroscopic properties of which reflect the polarity of their environment. These studies have most often used l-amino-8-naphthalene sulphonate (ANS) as a probe, but a more suitable probe, in principle, is 6-propionyl 2-(N,N-dimethyl)aminonaphthalene (PRODAN)8. We have synthesized a molecule with the advantageous spectroscopic properties of PRODAN but with a higher affinity for apomyo-globlin: 2′-(N,N-dimenthyl)amino-6-naphthopyl-4-trans-cyclo-hexanoic acid (DANCA), and report here its use to determine the polarity of the myoglobin haem pocket. Our results show that the pocket is actually a polar environment, and the polarity can be accounted for by peptide amide dipoles.

Theory of differential phase fluorometry: Detection of anisotropic molecular rotations

A general description of transformations in the excited state is employed to derive the general equations for the differential delay and the modulation ratio of the fluorescence owing to different pairs of excited species present. These equations yield directly the differential delay and the modulation ratio of the polarized components of the fluorescence from a rotating spherical molecule. Similar equations for a rotating irregular molecule are then derived from the sine and cosine transforms of the impulse response of the polarized components of the fluorescence emission. It is shown that for excitation at a wavelength at which the limiting polarization is high, namely, 1/2 to 3/11, the maximum differential tangent observed for anisotropic rotations is uniformly lower than the value for the sphere. The magnitude of this tangent defect permits an estimate of the minimum standard deviation of the principal rotational rates of the ellipsoid of inertia that describes the rotations of the molecule.

Absolute measurements of fluorescence polarization at high pressures

The measurement of polarization of the fluorescence of solutions under high pressure is rendered uncertain by the photoelastic birefringency of the windows, and needs a correction dependent upon the fraction of the light emerging from a window with polarization normal to that of the incident light (scrambling coefficient). This correction, which increases rapidly with applied pressure, differs according to whether the method of measurement employs rotation of the excitation polarizer (T format) or the emission polarizer (L format), and the latter is shown to involve the more reliable and the smaller correction. A four window pressure bomb housing a 1 ml volume cuvette that permits absorption and fluorescence measurements up to 4 kbar is described. The polarization correction up to 2.5 kbar was determined. Its reliability was demonstrated by measurements of the fluorescence polarization spectra of fluorophores in solvents of high viscosity at all pressures and by comparison of isopiestic and isothermal mea...

Tryptophan emission from human hemoglobin and its isolated subunits.

Abstract— The emission spectra of human adult hemoglobin A0 and its isolated α and ß subunits were obtained using a highly sensitive photon‐counting spectrofluorometer. The quantum yields of the emissions, relative to free tryptophan, were also measured as well as the excitation polarization spectra for hemoglobin A0 and apohemoglobin. The fluorophore bis‐ANS was utilized to probe for the presence of apoproteins in the hemoprotein preparations. The work suggests that tryptophan may be useful as an intrinsic probe to study dynamical processes in hemoglobin.