ISOLATION and SPECTROSCOPIC CHARACTERIZATION OF THE B875 ANTENNA COMPLEX OF A MUTANT OF Rhodopseudomonas sphaeroides

Abstract— We describe a procedure of purification of the B875 antenna complex isolated from the 3P17 mutant strain of Rhodopseudomonas sphaeroides, enriched in B875. The integrity of this isolated complex, as well as a very low content of residual B800-850 antenna, was suggested from low temperature absorption and resonance Raman spectra. Time resolved experiments were also carried out. The important result is the identity of the fluorescence lifetime of the B875 isolated complex (0.64 ± 0.03 ns) with that of the B875 antenna in vivo (0.63 ns), in the membrane of the C71 reaction center-less mutant of Rhodopseudomonas sphaeroides, measured in our previous study.
Our data suggest that the interactions between the bacteriochlorophylls of the complex, as well as the constraints imposed by their protein environment are not much changed from the in vivo state.     

PICOSECOND FLUORESCENCE KINETICS and ENERGY TRANSFER IN THE ANTENNA CHLOROPHYLLS OF GREEN ALGAE*

Single-photon timing measurements on flowing samples of Chlorella vulgaris and Chlamydomonas reinhardtii at low excitation intensities at room temperature indicate two main kinetic components of the fluorescence at open reaction centers (F₀) of photosystem II with lifetimes of approx. 130 and 500 ps and relative yields of about 30 and 70%. Closing the reaction centers progressively by preincubation of the algae with increasing concentrations of 3-(3′,4′-dichlorophenyl)-l,l-dimethylurea (DCMU) and hydroxylamine gave rise to a slow component with a lifetime increasing from 1.4 to 2.2 ns (F_max) The yield of the slow component increased to 65-68% of the total fluorescence yield in parallel to a decrease in the yield of the fast component to a value close to zero at the f_max-level. The 130 ps lifetime of the fast component remained unchanged. The middle component showed an increase of its lifetime from 500 to 1100 ps and of its yield by a factor of 1.5. Spacing of the ps laser pulses by 12 μs allowed us to resolve a new long-lived fluorescence component of very small amplitude which is ascribed to a small amount of chlorophyll not connected to functional antennae. The opposite dependence of the yield of the fast and the slow component on the state of the reaction centers at almost constant lifetimes is consistent with a mechanism of energy conversion in largely separately functioning photosystem II units. Yields and lifetimes of these two components are in agreement with the high quantum yield of photosynthesis. The lower lifetime limit of 1.4 ns of the slow component is assigned to the average transfer time of an excited state from a closed to a neighboring open reaction center and the increase in the lifetime to 2.2 ns is evidence for a limited energy transfer between photosystems II. Relative effects of changing the excitation wavelength from 630 to 652 nm on the relative fluorescence yields of the kinetic components were studied at the fluorescence wavelengths 682, 703 and 730 nm. Our data indicate that (i) the middle component has its fluorescence maximum at shorter wavelength than the fast component and (ii) that the antennae chlorophylls giving rise to the middle component are preferentially excited by 652 nm light. It is concluded that the middle component originates from the light-harvesting chlorophyll alb protein complexes and the major portion of the fast component from the chlorophyll a antennae of open photosystem II reaction centers.     

FLUORESCENCE AND THE LOCATION OF TRYPTOPHAN RESIDUES IN PROTEIN MOLECULES

Abstract— Fluorescence spectra of a number of native and denaturated proteins have been analysed, using spectral band width (ΔΛ), spectral maximum position (Λ_m), fluorescence quenching by external ionic quenchers, lifetime (b), and quantum yield ( q ) and its changes upon denaturation. The results enabled a model of fluorescence properties of tryptophan residues in the proteins to be substantiated by considering the existence of three discrete spectral classes, one buried in nonpolar regions of the protein (Λ_m 330–332 nm, ΔΛ= 48–49nm, q 0.11, τ= 2.1 ns) and two on the surface. One of the latter is completely exposed to water (Λ_m# 350–353 nm, ΔΛ= 59–61 nm, q # 0.2, τ= 5.4 ns); the other is in limited contact with water which is probably immobilized by bonding at the macromolecular surface (Λ_m# 340–342 nm, ΔΛ= 53–55 nm, q # 0.3, = 4.4 ns). Some quantitative predictions from the model, for (a) the fraction of fluorescence that is quenched by ionic quenchers, (b) the mean values of quantum yield, and (c) the mean values of fluorescence lifetime for various proteins, show good concordance with independent experimentally determined values.     

PICOSECOND FLUORESCENCE STUDY OF PHOTOSYNTHETIC MUTANTS OF Chlamydomonas reinhardii: ORIGIN OF THE FLUORESCENCE DECAY KINETICS OF CHLOROPLASTS

Abstract— The fluorescence decay kinetics of photosynthetic mutants of Chlamydomonas reinhardii which lack photosystem II (PS II), photosystem I (PS I), and both PS II and PS I have been measured. The PS II mutant strain8–36C exhibits fluorescence decay lifetime components of 53, 424 and 2197 ps. The fluorescence decay of a PS I mutant strain12–7 contains two major fluorescence decay components with lifetimes of 152 and 424 ps. The fluorescence decay of mutant strain C2, which lacks both PS II and PS I, is nearly single exponential with a lifetime of 2561 ± 222 ps. In simulations in which it is assumed that wild-type decays are a simple sum of the major decay components of the isolated parts of the photosynthetic unit as measured in the mutants, curves are obtained that fit the wild-type C. reinhardii fluorescence decay data when the absorption cross-sections of PS II and PS I are weighted approximately equally. The 89 ps lifetime component in the wild-type is an average of 53 and 152 ps components arising from excitation transfer to and trapping in PS I and PS II. The single step transfer time in PS I is estimated to be between 100 and 700 fs depending on assumptions about array size. We find that between two and four visits to the PS I reaction center are required before final trapping.     

Lifetimes of bacteriochlorophyll fluorescence in Rhodopseudomonas viridis and Heliobacterium chlorum at low temperatures

Fluorescence lifetimes of isolated membranes of Rhodopseudomonas viridis were measured in the temperature range of 77 K to 25 K. At room temperature, the main component of the fluorescence decay of bacteriochlorophyll (BChl) b had a time constant of 50 ps. In contrast to other purple bacteria, the emission at low temperature was spectrally homogeneous and showed essentially single lifetimes of 140 ps at 77 K and 180 ps at 25 K, with the primary electron donor in the oxidized state. Taking into account the relative fluorescence yields with open and closed reaction centers, we arrive at numbers of 125 ps and 215 ps, respectively, for open reaction centers. These numbers are significantly smaller than expected on the basis of measurements of the efficiency of charge separation, perhaps suggesting that the excitation decay in the absence of reaction centers is considerably faster at low temperature than at room temperature. At least four different spectral components with different lifetimes were observed at 25 K in the emission of Heliobacterium chlorum, a short-wavelength component of about 30 ps and three longer-wavelength components of about 100 ps, 300 ps, and 900 ps. This indicates a strong heterogeneity in the emitting pigment, BChl g-808. The component with the shortest lifetime does not appear to be affected by the redox state of the reaction center and might reflect energy transfer to BChl g species which are connected to the reaction center.     

QUENCHING OF THE FLUORESCENCE OF 8-METHOXYPSORALEN BY PROTONS

Abstract— The quenching of 8-methoxypsoralen (8-MOP) fluorescence by protons was observed to occur at the diffusion controlled rates in aqueous solutions at room temperature. Enhanced basicity of 8-MOP in the excited state compared to the ground state is expected on theoretical grounds. The fluorescence yield. which we determined as 6.3 × 10^--⁴ at pH 1 is surprisingly low and indicative of extremely fast radiationless decay pathways. The fluorescence lifetime of 8-MOP in neutral aqueous solution is on the order of 1–2 ns.     

FLUORESCENCE KINETICS OF SPINACH CHLOROPLASTS MEASURED WITH A PICOSECOND OPTICAL KERR GATE

Abstract. An overview of the reported chlorophyll a fluorescence lifetimes from green plant photosystems is presented and the problems encountered in the measurement of fluorescence lifetime using two currently available picosecond techniques are discussed.
The fluorescence intensity of spinach chloroplasts exposed to 10 ps flashes was measured as a function of time after the flash and wavelength of observation by the ultrafast Kerr shutter technique. Using a train of 100 pulses separated by 6ns and with an average photon flux per pulse of ˜2 times 10¹⁴ photons/cm², the fluorescence intensity at 685 nm (room temperature) was found to decay with two components, a fast one with a 56 ps lifetime, and a slow one with a 220 ps lifetime. The 730 nm fluorescence intensity at room temperature decays as a single exponential with a 100 ps lifetime. The 730 nm fluorescence lifetime was found to increase by a factor of 6 when the temperature was lowered from room temperature to 90 K while the lifetime of 685 and 695 nm fluorescence were unchanged. At room temperature, the fast and slow components at 685 nm are attributed to the emission from pigment system I (PS I) and PS II, respectively. It is likely that the absolute values of lifetimes, reported here, may increase if single ps low intensity flashes are used for these measurements.     

MULTIPLE EXCITATIONS AND THE YIELD OF CHLOROPHYLL a FLUORESCENCE IN PHOTOSYNTHETIC SYSTEMS

Abstract. Analysis of the effect of multiple excitations on chlorophyll a fluorescence yields in the green alga Chlorella reveals several distinct reactions. The first excitation in dark-z-adapted units produces photochemistry and the high yield state with a rise time of 35 ns. It is ascribed to a change in coupling between the antenna pigments and the photochemical trap. The second hit produces with the same quantum yield a quenched state which changes to the high yield state with a rise time of 4 μ s. This is ascribed to the formation and the decay of a particular carotenoid triplet state near the funnel or antenna-trap junction. Further hits produce enhanced quenching assigned to mobile triplets with lifetimes in the order of 100 ns. The fluorescence yield decreases monotonically with increasing excitations during the 7 ns pulse. This effect can be adequately ascribed to annihilation of excitations with lifetimes longer than the trapping time, or by a unique model of a multi-trapped unit. The latter model is favored by arguments based both on the absence of a local maximum in the graph of fluorescence yield vs excitation energy and on the fact that the high yield state shows a different behaviour on multiple excitation, fit by a single-trapped unit. This analysis is related to that used in experiments with ps flashes and is applied to the qualitatively different bacterial system.     

Effect of Supplementary UVB Radiation on Chlorophyll Synthesis and Accumulation of Photosystems during Chloroplast Development in Spirodela oligorrhiza

Abstract— Although the effects of ultraviolet B (UVB, 290–320 nm) radiation have been studied in plants extensively, little is known about the potential impacts on maturation of chloroplasts. To address this problem, the effects of supplementary UVB on chloroplast development were examined in the aquatic higher plant Spirodela oligorrhiza. Dark-grown Spirodela-containing proplastids were exposed to photosynthetically active radiation (PAR) and ultraviolet A (UVA, 320–400 nm), plus supplementary UVB equivalent to 1% of PAR on a photon basis. The biosynthesis and assembly of chlorophyll (Chi) into reaction centers was followed for 4 days in situ by low temperature (77 K) Chi fluorescence. Impacts on chloroplast development were detected after only 1 h incubation in light with supplementary UVB. Fluorescence emission signals from Chi associated with the photosystem (PS) II antenna, PSII reaction centers and PSI reaction centers were detected at the same time with or without UVB, but the magnitude of PS fluorescence was diminished up to 60% in plants incubated in UVB. The Chi content was also lower in UVB-treated plants, but to a lesser degree than anticipated by low temperature fluorescence, suggesting lack of organization and/or association of Chi with PS. Electron transport, measured with room temperature fluorescence induction, was not consistently different in plants exposed to UVB. These results suggest that with UVB, fewer and/or smaller PS form during chloroplast development, but there is not a large inhibition of Chi synthesis or PSII activity.     

Comparative Time-Resolved Photosystem II Chlorophyll a Fluorescence Analyses Reveal Distinctive Differences between Photoinhibitory Reaction Center Damage and Xanthophyll Cycle-Dependent Energy Dissipation*

Abstract— The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important pho-toprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (ΔpH) and the carotenoid pigment interconversions of the xanthophyll cycle. Co-incidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chi a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chi a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhi-bition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from ～2 ns to ～0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonra-diative decay path for the charge-separated state of the PSII reaction center. In contrast, the ΔpH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et ah, 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the ΔpH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.     

新型双发色团染料荧光光谱及其寿命的研究

有效的染料激光操作需要较高的荧光量子效率,若丹明是在500~700 nm光谱区中一类最重要的激光染料.然而,染料的基态分子和三线态对辐射能量的吸收将会大大降低激光输出效率,再者,由于若丹明类染料在紫外区的吸收系数较小,为了有效吸收泵浦能量(如用XeCI准分子激光,308 nm),就必须使用高浓度染料溶液,在这种情况下,若丹明类染料较小的Stokes位移就势必造成基态分子更大的重复吸收,即造成更大的谐振腔损耗^[1].     

LINEAR AND CIRCULAR DICHROISM AND FLUORESCENCE POLARIZATION OF THE B875 LIGHT-HARVESTING BACTERIOCHLOROPHYLL-PROTEIN COMPLEX FROM RHODOPSEUDOMONAS SPHAEROIDES

Abstract— The orientation of the chromophores in the B875 light-harvesting bacteriochlorophyll complex isolated from Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis was examined by linear and circular dichroism and fluorescence polarization procedures. The circular dichroism in the near-IR was weaker than that of the B800–850 light-harvesting complex and had a distinctly different shape. This suggested a different geometry for the two bacteriochlorophylls of B875 and less interactive association between their transition moments. The magnitude of the circular dichroism in the carotenoid region of B875 was similar to that of B800–850 but gave more negative values between approx. 430–485 nm; this may reflect a difference in the asymmetric binding of carotenoids to the B875 protein. The fluorescence polarization increased sharply across the near-IR region of B875 and achieved very high values at long wavelengths. This confirmed that more than one transition contributed to this absorption band. The linear dichroism of B875 did not show a significant change in this near-IR band like that observed for the longest wavelength band of B800–850. Thus, the transition moments for each bacteriochlorophyll within B875 appear to be tilted to approximately the same extent with respect to the protein axis. These results distinguish B875 from all other light-harvesting complexes and suggest that the antennae of Rhodospirillaceae which contain a single near-IR absorption band cannot be classified into a single group.     

GLOBAL ANALYSIS OF THE TIME-RESOL VED FLUORESCENCE OF α-CHYMOTRYPSINOGEN A AND α-CHYMOTRYPSIN POWDERS AS A FUNCTION OF HYDRATION

The time-resolved tryptophyl fluorescence of alpha-chymotrypsin A and alpha-chymotrypsin in the crystalline state and in buffer solution at room temperature was analyzed globally. Triple-exponential decay functions are necessary to adequately describe the tryptophyl fluorescence decay surfaces of the protein powders as a function of hydration and in solution. The fluorescence lifetimes of alpha-chymotrypsinogen A (tau 1 = 0.32, tau 2 = 1.30 ns, tau 3 = 3.98 ns) and alpha-chymotrypsin(tau 1 = 0.66 n s, tau 2 = 2.26 ns, tau 3 = 5.40 ns) are constant over the entire hydration range. The spectral positions of the decay-associated spectra of the hydrated powders do not shift as a function of hydration. This indicates that the structures of the zymogen and the active enzyme are unaffected by hydration. The lifetimes of alpha-chymotrypsinogen A in phosphate buffer pH 7.4 are tau 1 = 0.37 ns, tau 2 = 1.17 ns and tau 3 = 3.44 ns while the respective values of alpha-chymotrypsin are tau 1 = 0.47 ns, tau 2 = 1.40 and tau 1 = 3.89 ns.     

NONLINEAR POLARIZATION SPECTROSCOPY (FREQUENCY DOMAIN) STUDIES OF EXCITED STATE PROCESSES: THE B800–850ANTENNA OF RHODOBACTER SPHAEROIDES

Nonlinear polarization spectroscopy in the frequency domain allows rate constant determinations of fast electronic energy and phase relaxations together with characterization of the type of line broadening. Application of this method to the B850 component of the isolated B800–850antenna ofRhodobacter sphaeroides at room temperature shows that B850 is inhomogeneously broadened, with homogeneous widths between 30 and 200 cm^?1, depending on the spectral position of the subforms. The corresponding phase relaxation times are clearly in the subpicosecond range. There is also indication of an up-to-now unspecified1–5 ps energy relaxation channel per subunit.     

Isomers in the excited state of electron-transferring flavoprotein from Megasphaera elsdenii: spectral resolution from the time-resolved fluorescence spectra

Electron-transferring flavoprotein (Holo-ETF) from Megasphaera elsdenii contains two FAD's, one of which easily dissociates to form Iso-ETF (contains one FAD). Time-resolved fluorescence of FAD in Iso-ETF, and Holo-ETF were measured at 5 degrees C and 25 degrees C. Wavelength-dependent fluorescence decays of the both ETF at 5 degrees C and 25 degrees C were analyzed to resolve them into two independent spectra. It was found that Iso-ETF displayed two spectra with lifetime of 0.605 ns (emission peak, 508 nm) and with lifetime of 1.70 ns (emission peak, 540 nm) at 5 degrees C, and with lifetime of 0.693 ns (emission peak, 508 nm) and with lifetime of 2.75 ns (emission peak, 540 nm) at 25 degrees C. Holo-ETF displayed two spectra with lifetime of 0.739 ns (emission peak, 508 nm) and with lifetime of 2.06 ns (emission peak, 545 nm) at 5 degrees C, and with lifetime of 0.711 ns (emission peak, 527 nm) and with lifetime of 3.08 ns (emission peak, 540 nm) at 25 degrees C. Thus fluorescence lifetimes of every spectrum increased upon elevating temperature. Emission peaks Iso-ETF did not change much upon elevating temperature. Activation enthalpy changes, activation entropy changes and activation Gibbs energy changes of quenching rates all displayed negative. Two emission species in the both ETF may be hydrogen-bonding isomers, because isoalloxazine ring of FAD contains four hydrogen acceptors and one donor.     

MOLECULAR LUMINESCENCE OF SOME ISOALLOXAZINES IN APOLAR SOLVENTS AT VARIOUS TEMPERATURES

Abstract— –Spectral properties of isoalloxazines in organic solvents of low polarity are determined at 300 and 77 K. Vibrational structure in the spectra reveals a vibrational mode of 1250cm^-. The pure electronic transition energies are established to a greater accuracy than was done previously and comparison to theoretical data is made. Actual lifetimes up to 10 ns for fluorescence and 300 ms for phosphorescence are found. The ratio of the actual fluorescence lifetime and the radiative lifetime is found to agree well with the quantum yield. Solvent interactions hardly shift the energy of the first electronically excited singlet state but merely affect the Franck-Condon envelope of the spectrum and the non radiative decay of the chromophore. In albne solutions at 77 K isoalloxazine clusters are formed exhibiting P-type delayed fluorescence.     

NANOSECOND LASER PHOTOLYSIS OF PHOBORHODOPSIN: FROM Natronobacterium pharaonis APPEARANCE OF KL AND L INTERMEDIATES IN THE PHOTOCYCLE AT ROOM TEMPERATURE

Abstract— Photochemical and subsequent thermal reactions of pharaonis phoborhodopsin (ppR; absorption maximum, 498 nm) from Natronobacrerium pharaonis were investigated by nanosecond laser photolysis at 20°C. The experimental results clearly showed the presence of two intermediates in the photocycle of ppR besides the K, M and O intermediates detected previously. One was formed immediately after the excitation of ppR with a blue pulse (pulse width, 17 ns; wavelength, 460 nm), and the other was formed by the thermal reaction of this species. The new intermediates' absorption maxima were 512 and 488 nm, their extinction coefficients were 0.85- and 0.68-times smaller than that of ppR, and their lifetimes were 990 ns and 32 μs, respectively. The absorption and kinetic characteristics of these intermediates relative to ppR were similar to those of the KL and L intermediates of bacteriorhodopsin (bR). The formation of KL intermediates from both ppR and bR were observed only at room temperatures. On the other hand, the formation of L intermediate of bR was observed at both of room and low temperature, whereas that from ppR only at room temperature. The unique formation of L intermediate of ppR at room temperature is discussed in relation to high thermal stability of K intermediate of ppR.     

Assembly of Photosystem I and II during the Early Phases of Light-Induced Development of Chloroplasts from Proplastids in Spirodela oligorrhiza

The aquatic higher plant Spirodela oligorrhiza , which contains proplastids when grown in the dark, was used to study light-dependent chloroplast development. Low-temperature (77 K) and room temperature fluorescence were utilized in situ on whole plants to examine plastid development. The dark-grown plants contain two 77 K fluorescence peaks, at 633 nm (F633) and at 657 nm (F657), with F633 dominating. The F657 species represents protochlorophyllide that is bound to protochloro-phyllide oxidoreductase. It was rapidly phototrans-formed to chlorophyllide (within 5 s) via a monomolec-ular reaction. Free protochlorophyllide (F633) was converted to chlorophyllide during a 3 h exposure to light. Photosystem (PS) assembly in Spirodela could be detected 2 h after the plants were first exposed to light, with the PSII reaction center (77 K fluorescence at 684 nm) appearing slightly before the PSI reaction center (77 K fluorescence at 725 nm). After the first reaction centers were formed the antenna complexes were added; the light-harvesting complex (LHC) I of PSI appeared after 8 h, and 47 kDa chlorophyll protein of PSII appeared between 12 h and 24 h. After 30 h of exposure to light, the plants acquired the ability to perform a light state transition, marking the appearance of functional LHCII complexes in the developing chloroplast. Finally, it was found that photosynthetic activity, as measured by room temperature chlorophyll fluorescence, accelerated con-comitantly with detection of the antenna complexes. Therefore, although reaction centers are detected very early during the proplastid to chloroplast conversion, they may have little activity or be unstable until the antennae are present.     

A HIGH RESOLUTION FLUORESCENCE DECAY AND DEPOLARIZATION STUDY OF HUMAN PLASMA APOLIPOPROTEINS

Abstract— Human plasma apolipoprotein A-I (apoA-I) and apolipoprotein C-I (apoC-I) were investigated by time-resolved fluorescence decay and depolarization. The tryptophyl fluorescence of apoA-I undergoes a double-exponential decay with lifetimes of 1.07 and 3.43 ns which remain unchanged over the range of apoA-I concentration studied.
The time-resolved fluorescence of both native and denatured forms of apoC-I exhibits an unusual tryptophyl fluorescence decay that was best fit to a triexponential function with lifetimes at 3.7 ± 0.2, 1.1 ± 0.1 and 0.1 ns at 2°C. The native and denatured forms of apoC-I had rotational correlation times of 1.42 and 1.19 ns at 20°C respectively. A shorter rotational correlation time associated with the internal tryptophan motions was not observed or resolved.
The decay of tryptophyl fluorescence in apoC-I/DPPC/cholesterol complex at 20°C is also triexponential with lifetimes at 4.94, 1.28 and 0.21 ns, which are longer than those of the uncomplexed forms. Two rotational correlation times of 28.32 and 0.59 ns at 20°C were resolved by fluorescence depolarization measurements. The long rotational time remained constant with temperatures above 30°C. Also, the temperature dependence of the order parameter, S², resembled a lipid phase transition curve with a transition midpoint at 38°C. The tryptophan and thus apoC-I are found to be affected by the bulk changes in the lipid.     

STUDY ON THE PROTONATION MECHANISM OF 2,6-DIMETHYL NAPHTHALENE DICARBOXYLATE

By the study of absorption and fluorescence spectra and the lifetime of fluorescence at room and low temperatures of 2,6-dimethyl naphthalene dicarboxylate (DMN) in different concentrations of sulfuric acid, different interactions between molecules of DMN and sulfuric acid have been observed. These interactions have been revealed by the absorption spectra of charge transfer complex in the ground state, emission of exciplex, absorption spectra of hydrogen bonding interaction, absorption and emission spectra after proton transfer and different lifetimes before and after protonation. The interaction mechanism of DMN and sulfuric acid through first the CT complex and exciplex then hydrogen bonding and finally proton transfer is proposed.