Ari Gafni

Sensitive Instrument for the Study of Circular Polarization of Luminescence

A sensitive instrument is described for the measurement of the extent of circular polarization of light emitted by chiral molecules. In this instrument the intensity of the circularly polarized component of the luminescence light is selectively modulated by an electro‐optic or photoelastic light modulator, while the unpolarized component of the luminescence is unaffected. The modulated light beam is detected by a photomultiplier and the ac component of the electric signal produced is amplified by a phase‐sensitive amplifier tuned to the modulation frequency. The light used for the excitation of the sample and the luminescence light are monochromated. An accessory is described which produces controlled mixtures of unpolarized and circularly polarized light and thus permits calibration of the output of the instrument in terms of the degree of circular polarization of the light under examination. Signal to noise ratios of 20 have been readily obtained with luminescence which is circularly polarized to an extent of 10−3 to 10−4.

Excited state proton transfer reactions of acridine studied by nanosecond fluorometry

Abstract Rate constants for excited state protonation reactions of acridine in aqueous solutions have been determined using fluorescence decay measurements. The value of the excited state pK derived from the rate constants is in full agreement with the one obtained from steady state fluorescence measurements. It is concluded that acridine follows a two states reaction scheme in its electronically excited state.

Age-related changes in the redox status of rat muscle cels and their role in enzyme-aging

The redox status of three biological components capable of undergoing oxidation-reduction reactions, glutathione, NAD and NADP, were determined in muscle tissues of young and old rats. A considerable increase in the relative concentration of the oxidized form, at the expense of the reduced one was found in the old tissue reflecting a significantly less reducing environment than in young cells. The effects of varying the ratio of reduced/oxidized glutathione in vitro on the activity of the enzyme glyceraldehyde-3-phosphate dehydrogenase extracted from young and old animals were compared. It was found that concentrations of GSSG as found in old muscle tissue do not affect enzyme samples extracted from young muscle. The accumulation of oxidized glutathione observed in old cells does not, therefore, directly cause the age-related activity loss of this enzyme.

Circular polarization of fluorescence of chlorophyll in solution and in native structures

Chlorophyll dimers in solution, subchlorplast particles and chloroplasts were investigated by their circular dichroism and circular polarization of their fluorescence, which reflect their optical rotatory power in the ground state and electronically excited state, respectively. The chlorophyll dimers in fluid solution lose their optical activity upon electronic excitation, reflecting a marked concomitant change in the structure of the dimers. This change is arrested in a solution of very high viscosity. The pronounced difference between the circular polarization of the dimers in fluid media and that of subchloroplast particles and chloroplasts indicates that the former are not suitable models for associated chlorophyll in native structures in electronically excited states. Impairment of the photochemical activity of chloroplasts by heat treatment is accompanied by a reduction of the circular polarization of the fluorescence, which probably reflects a disorganization in structure. The same extent of circular polarization was observed in the fluorescence of chloroplasts regardless whether the reaction centers are open or closed; thus either the same molecules are emitting in the two cases or, if different molecules emit, they are packed in a similar way.

The interaction of adenine with its binding site in rabbit muscle glyceraldehyde 3-phosphate dehydrogenase studied by fluorescence decay

Abstract The fluorescence decay mechanism of 1, N6-ethenoadenosine diphosphoribose bound to rabbit muscle glyceraldehyde 3-phosphate dehydrogenase markedly differs from that of the intact coenzyme analog (eNAD+) bound to the same enzyme. In the latter case the fluorescence is partially quenched by interactions between the ethenoadenine ring and amino acid residues in its binding site. Binding of the nicotinamide moiety of the coenzyme thus affects the relative orientation of the adenine ring within its binding site leading to the quenching interactions. The interactions of the adenine group with its binding site induce conformational changes in the enzyme which affect the binding of additional coenzyme molecules. The nicotinamide base thus determines, indirectly, the negative cooperativity found in NAD+ binding.

Age-Related Effects on Subunit Interactions in Rat Muscle Glyceraldehyde-3-phosphate Dehydrogenase

Publisher Summary This chapter describes age-related effects on subunit interactions in rat muscle glyceraldehyde-3-phosphate dehydrogenase. It presents a study that compares the dissociation patterns of L-glyceraldehyde-3-phosphate dehydrogenase (GPDH) from young and old rat muscle. The effects induced in the dissociation mechanism by adenosine diphosphate (ADP) are also presented and analyzed to characterize the structural modifications induced in the old enzyme molecule. The dependence of enzymatic activity on GPDH concentration was studied by making a series of dilutions, from one stock solution of concentrated enzyme, thus preparing samples at various concentrations. These were incubated for 30 minutes at the desired temperature and assayed. Inactivation in presence of ADP was studied in a similar way as a function of both GPDH and the nucleotide concentrations. The determination of concentrations and assays of enzymatic activity was performed on a Zeiss Model PMQ II spectrophotometer. All measurements were done at 22 °C. The concentration-dependent inactivation of rat muscle GPDH fully agrees with the assumption that equilibrium exists between the active tetramer and a completely inactive dimer. The dissociation constants calculated from the results of the study show the old enzyme form to be more dissociable than the young species. This finding strongly supports the results of sedimentation velocity experiments in which the old enzyme form was shown to dissociate more readily, in the presence of sodium chloride, than its young counterpart.

6-Aminohexanoic acid-plasminogen interactions studied by fluorescence.

Abstract Circular polarization of luminescence spectra of human plasminogen and of its derivatives were measured in solutions of ligand-free proteins and with saturating amounts of 6-aminohexanoic acid. Spectroscopic changes induced by the ligand reveal similar perturbations of the binding sites in all the protein derivatives. It is concluded that the gross conformational change induced by 6-aminohexanoic acid binding to the native plasminogen involves changes of sterical relations of entire protein domains.

Apparent microviscosity of intact and post-lipolysis (“remnant”) very low density lipoprotein particles

The apparent microviscosity of intact rat plasma very low density lipoprotein (VLDL) and post-lipolysis very low density lipoprotein was determined by fluorescence depolarization measurements and flurorescence decay measurements using 1, 6-diphenylhexatriene. Post-lipolysis very low density lipoprotein was prepared in vitro after incubation of the intact lipoprotein with either purified bovine milk lipoprotein lipase or lipoprotein lipase rich (post-heparin) plasma. During lipolysis, an average of 88% of the triglycerides were hydrolyzed, and the lipoprotein became depleted in phospholipids, cholesterol and apolipoprotein C. The apparent microviscosity of the lipoprotein increased by three-fold from 0.63 to 1.88 poise. It is concluded that the compositional changes occurring during lipolysis affect the physical properties of the lipoprotein, as measured here by the fluidity (microviscosity) of the particles.

The interaction of liver alcohol dehydrogenase with NADH as studied by differential protein denaturation

Heat denaturation of horse liver alcohol dehydrogenase was followed in the presence of isobutyramide at various degrees of saturation of the binding sites by NADH. A study of the fluorescence enhancement which is observed when an excess of NADH is added to the partially denatured mixtures provides information regarding the relative concentrations of mono- and bioccupied enzyme molecules. This approach is of value in situations when the association constants for coenzyme are so large that the concentration of the free ligand is negligible. The results obtained indicate that the binding of NADH to liver alcohol dehydrogenase follows the statistically predicted distribution. At the same time evidence was obtained for interaction between the two subunits of the enzyme.