Time-resolved natural and magnetic circular dichroism measurements on the nanosecond to second time scale have been used to investigate many chemical and biological processes. The key element that makes these measurements possible is a simple and inexpensive variable linear retarder (a strain plate) which produces well-characterized, elliptically polarized light throughout a broad spectral range (180–1500 nm). It essentially consists of a fused silica plate of high optical quality appropriately compressed from the edges to produce ∼1° of retardation. Early designs that consisted of a square plate compressed with wedges have worked satisfactorily in the past, but their use was complicated by inhomogeneity of strain across the probe beam (as large as 6 mm diameter for white light measurements with a flashlamp) and a temperature dependence of the strain. An improved strain plate that consists of a round fused silica plate compressed with springs is presented, and its performance is evaluated with respect to earlier designs. This improved strain plate not only simplifies time-resolved circular dichroism measurements but also improves the quality of the data and the kinetic resolution of intermediates.

Nanosecond time-resolved absorption measurements are reported for the room temperature photolysis of a modified rhodopsin pigment, 13-demethylrhodopsin, which contains the chromophore 13-demethylretinal. The measurements are consistent with the formation of an equilibrium between a BA-THO-13-demethylrhodopsin species and a blue-shifted species (relative to the parent pigment), BSI-13-demethylrhodopsin. The results are compared to those acquired after photolysis of native bovine rhodopsin [Hug, S. J., Lewis, J. W., Einterz, C. M., Thorgeirsson, T. E., & Kliger, D. S. (1990) Biochemistry (preceding paper in this issue)] and to results obtained after photolysis of several modified isorhodopsin pigments in which the BSI species was first observed. It is concluded that in all of the pigments the results are consistent with the formation of an equilibrium between BATHO and BSI, which subsequently decays on a nanosecond time scale at room temperature to a lumirhodopsin intermediate.

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.

The photolysis intermediates of an artificial bovine rhodopsin pigment, cis-5,6-dihydro-isorhodopsin (cis-5,6,-diH-ISORHO, lambda max 461 nm), which contains a cis-5,6-dihydro-9-cis-retinal chromophore, are investigated by room temperature, nanosecond laser photolysis, and low temperature irradiation studies. The observations are discussed both in terms of low temperature experiments of Yoshizawa and co-workers on trans-5,6-diH-ISORHO (Yoshizawa, T., Y. Shichida, and S. Matuoka. 1984. Vision Res. 24: 1455-1463), and in relation to the photolysis intermediates of native bovine rhodopsin (RHO). It is suggested that in 5,6-diH-ISORHO, a primary bathorhodopsin intermediate analogous to the bathorhodopsin intermediate (BATHO) of the native pigment, rapidly converts to a blue-shifted intermediate (BSI, lambda max 430 nm) which is not observed after photolysis of native rhodopsin. The analogs from lumirhodopsin (LUMI) to meta-II rhodopsin (META-II) are generated subsequent to BSI, similar to their generation from BATHO in the native pigment. It is proposed that the retinal chromophore in the bathorhodopsin stage of 5,6-diH-ISORHO is relieved of strain induced by the primary cis to trans isomerization by undergoing a geometrical rearrangement of the retinal. Such a rearrangement, which leads to BSI, would not take place so rapidly in the native pigment due to ring-protein interactions. In the native pigment, the strain in BATHO would be relieved only on a longer time scale, via a process with a rate determined by protein relaxation.

The linear dichroism spectrum of rhodopsin in sonicated bovine disk membranes was measured 30, 60, 170, and 600 ns after room temperature photolysis with a linearly polarized, 7-ns laser pulse (lambda = 355 or 477 nm). A global exponential fitting procedure based on singular value decomposition was used to fit the linear dichroism data to two exponential processes which differed spectrally from one another and whose lifetimes were 42 +/- 7 ns and 225 +/- 40 ns. These results are interpreted in terms of a sequential model where bathorhodopsin (BATHO, lambda max = 543 nm) decays toward equilibrium with a blue shifted intermediate (BSI, lambda max = 478 nm). BSI then decays to lumirhodopsin (LUMI, lambda max = 492 nm). It has been suggested that two bathorhodopsins decay in parallel to their products. However, a Monte Carlo simulation of partial photolysis of solid-state visual pigment samples shows that one mechanism which creates populations of BATHO having different photolysis rates at 77 K may not be responsible for the two decay rates reported here at room temperature. The angle between the cis band and 498-nm band transition dipoles of rhodopsin is determined to be 38 degrees. The angles between both these transition dipoles and those of the long-wave-length bands of BATHO, BSI, and LUMI are also determined. It is shown that when BATHO is formed its transition dipole moves away from the original cis band transition dipole direction. The transition dipole then moves roughly twice as much towards the original cis band direction when BSI appears. Production of LUMI is associated with return of the transition dipole almost to the original orientation relative to the cis band, but with some displacement normal to the plane which contains the previous motions. The correlation between the lambda max of an intermediate and its transition dipole direction is discussed.

Transient-absorption difference spectra from 320 nm to 700 nm were obtained at times ranging from 30 ns to 1200 ns after 532-nm photolysis of rhodopsin at room temperature. Kinetics on this time scale at various wavelengths are also presented. The isosbestic points between spectra acquired at successive times after photolysis shift from 510 nm to 530 nm. This shift is inconsistent with a simple process of one bathorhodopsin (BathoR) intermediate being transformed into one lumirhodopsin (LumiR) intermediate on this time scale. The kinetics at 425 nm, 515 nm, and 575 nm could not be fit well to a single-exponential expression. The data are consistent with the existence of two forms of BathoR (BathoR1 and BathoR2) that exhibit different spectra and decay kinetics. The BathoR1 absorption maximum lies near 565 nm, and the BathoR1/LumiR1 isosbestic point is near 430 nm. The BathoR2 absorption maximum lies near 535 nm, and the BathoR2/LumiR2 isosbestic point is near 480 nm. The kinetics at the isosbestic wavelengths were fit to single-exponential expressions corresponding to BathoR1 and BathoR2 lifetimes of 170 +/- 20 ns and 36 +/- 15 ns, respectively.

An optical multichannel analyzer system for obtaining high-quality spectra of intermediates formed by nanosecond laser photolysis is described. The system is useful for samples which undergo irreversible photochemistry and which are available in limited amounts. Sample volumes as small as 25 μl are possible and a syringe pump driven by a linear stepper motor is used to deliver fresh sample after each photolysis pulse. The microchannel-plate intensified detector used here in gated mode can collect an entire spectrum in 5 ns, but proper synchronization of the detector scans is required to avoid severe distortion which can appear as random noise in signal averaging. Noise from various sources such as phosphor lag in the detector, time jitter, source intensity fluctuations, and the photon character of light are discussed. Experimental design for noise reduction is described including diagnostic procedures and light source filtering to produce more uniform signal-to-noise ratio throughout the spectrum. Finally, a comparison is made between the noise observed to occur between detector channels and the noise predicted to occur based on the photoelectron gain of the intensifier. The disagreement between these is discussed in terms of the spatial resolution of the detector.

AbstractThe artificial 11‐cis‐retinal (I) (synthesis already published) forms a pigment when incubated with bovine opsin.

Chemischer InformationsdienstVolume 16, Issue 23 Physical Organic Chemistry ChemInform Abstract: NEW TECHNIQUE FOR MEASURING CIRCULAR DICHROISM CHANGES ON A NANOSECOND TIME SCALE. APPLICATION TO (CARBONMONOXY)MYOGLOBIN AND (CARBONMONOXY)HEMOGLOBIN J. W. LEWIS, J. W. LEWISSearch for more papers by this authorR. F. TILTON, R. F. TILTONSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorS. J. MILDER, S. J. MILDERSearch for more papers by this authorI. D. KUNTZ, I. D. KUNTZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author J. W. LEWIS, J. W. LEWISSearch for more papers by this authorR. F. TILTON, R. F. TILTONSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorS. J. MILDER, S. J. MILDERSearch for more papers by this authorI. D. KUNTZ, I. D. KUNTZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author First published: June 11, 1985 https://doi.org/10.1002/chin.198523051AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume16, Issue23June 11, 1985 RelatedInformation

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTBirefringence effects in transient circular dichroism measurements with applications to the photolysis of carbon monoxyhemoglobin and carbon monoxymyoglobinC. M. Einterz, J. W. Lewis, S. J. Milder, and David S. KligerCite this: J. Phys. Chem. 1985, 89, 18, 3845–3853Publication Date (Print):August 1, 1985Publication History Published online1 May 2002Published inissue 1 August 1985https://pubs.acs.org/doi/10.1021/j100264a015https://doi.org/10.1021/j100264a015research-articleACS PublicationsRequest reuse permissionsArticle Views105Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options Get e-Alerts

Nanosecond transient spectroscopic measurements of the BATHO products formed from photolysis of bovine rhodopsin (RHO) and isorhodopsin (ISO) are discussed. BATHO absorption spectra of RHO and ISO differ slightly, but both pigments exhibit wavelength maxima near 560 nm. An additional transient absorption at 440 nm is observed immediately following excitation of RHO but not ISO. The decay times and Arrhenius activation energies of the two 560 nm absorbing transients are the same. In addition to spectral differences in the photolysis of RHO and ISO, the bleaching yield as a function of 532 nm laser power is different, with the yield in RHO saturating at lower laser power than ISO. The bleaching yield of the two pigments has been modeled using the known extinction coefficients and quantum yields for the interconversion of RHO, ISO, and a single BATHO species. Agreement between experiment and the model is found if the effects of the laser polarization are considered. The data are consistent with a common BATHO in the photolysis of RHO and ISO.

Chemischer InformationsdienstVolume 14, Issue 18 Physical Organic Chemistry ChemInform Abstract: EVIDENCE THAT THE EXCITED-STATE GEOMETRY OF DIPHENYLBUTADIENE IS NEARLY PLANAR W. A. YEE, W. A. YEESearch for more papers by this authorJ. S. HORWITZ, J. S. HORWITZSearch for more papers by this authorR. A. GOLDBECK, R. A. GOLDBECKSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author W. A. YEE, W. A. YEESearch for more papers by this authorJ. S. HORWITZ, J. S. HORWITZSearch for more papers by this authorR. A. GOLDBECK, R. A. GOLDBECKSearch for more papers by this authorC. M. EINTERZ, C. M. EINTERZSearch for more papers by this authorD. S. KLIGER, D. S. KLIGERSearch for more papers by this author First published: May 3, 1983 https://doi.org/10.1002/chin.198318053AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume14, Issue18May 3, 1983 RelatedInformation