SPECTRAL PROPERTIES OF THE RHODOPSIN-SYSTEM OF THE CRAYFISH Astacus leptodactylus

Abstract— The absorption spectra of the membrane-bound and of the digitonin-solubilized visual pigment of crayfish Astacus leptodactylus were investigated by conventional spectrophotometry. A method was developed to isolate purified rhabdoms almost entirely free from screening pigments from a single retina. The quantity of isolated and purified rhabdoms from a single retina was sufficient to measure the absorption spectra of the visual pigment.
The absorption spectra of the chromoprotein system (R and M) show that both the membrane-bound and the digitonin-solubilized visual pigment isomers are stable at 0°C and pH 7.0. Rhodopsin and metarhodopsin are photoreversible under these conditions without any light-induced denaturation. The difference spectra for the chromoprotein isomers and those of different photostationary states yield maximal values for ΔE at 570 and 485 nm.
At neutral pH, 0°C, Λ_max of rhodopsin is 530 nm. Irradiation with light of Λ= 630 to 640 nm isomerizes rhodopsin nearly quantitatively to metarhodopsin with Λ_max, of 500 nm. The molar extinction coefficient of metarhodopsin is greater than that of rhodopsin by a factor of ˜ 1.41. each measured at its respective Λ_max Metarhodopsin can be isomerized to rhodopsin by irradiating at Λ > 630 nm. As the absorption spectra of the two chromoprotein isomers overlap, only part of the metarhodopsin can be reversed to rhodopsin. The maximal photoreversion can be achieved by irradiating at 460 nm. The stability of the digitonin-solubilized chromoprotein is remarkably dependent on temperature. Warming the digitonin extract of rhabdoms from 0 to 20 or 30°C caused a shift of the rhodopsin spectrum to shorter wavelengths (Λ_max= 485 nm) accompanied by a decrease of E_Λmax by about 30%.     

THE INVOLVEMENT OF WATER AT THE RETINAL BINDING SITE IN RHODOPSIN AND EARLY LIGHT-INDUCED INTRAMOLECULAR PROTON TRANSFER

Abstract Extensive dehydration of air-dried films of bovine rod outer segment membranes induces fully reversible changes in the absorption spectrum of rhodopsin, indicative of deprotonation of the retinylidene Schiff base in more than 50% of the rhodopsin molecules in the sample. This suggests that water is involved at the site of the Schiff base protonation in rhodopsin. In contrast, the spectrum of metarhodopsin I is resistant to similar dehydrating conditions, implying a significant difference in the mechanism for protonation in metarhodopsin I. The photochemistry of dehydrated membranes was also explored. Photoexcitation of deprotonated rhodopsin (λ_max 390 nm) induces a large bathochromic shift of the chromophore. The major photoproduct at room temperature was spectrally similar to metarhodopsin I (λ_max, 478 nm). These findings suggest that intramolecular proton transfer involving the Schiff base proton may occur in the earlier stages of the visual cycle, prior to or during the formation of metarhodopsin I.     

PHOTOCHEMICAL REACTION OF 13-CIS-BACTERIORHODOPSIN STUDIED BY LOW TEMPERATURE SPECTROPHOTOMETRY

Abstract— The photoreaction cycle of 13- cis -bacteriorhodopsin (13- cis -bR) was investigated by low temperature spectrophotometry using two different preparations; 13- cis -bR constituted from bacterioopsin and 13- cis -retinal, and dark-adapted bacteriorhodopsin (bR^D), which is an equi-molar mixture of 13- cis -bR and trans -bR.
By irradiation with 500 nm light at — 190°C, 13- cis -bR was converted to its batho-product, batho-13- cis -bR (batho-bR¹³), which is different from batho-product from trans -bR, batho-bR^t. On warming batho-bR¹³ to -5°C in the dark, it completely changed to trans -bR. We estimated the composition of 13- cis -bR and trans -bR in the warmed sample spectrophotometrically and then the absorption spectrum of batho-bR¹³ was calculated. The absorption maximum lies at 608 nm, 1250 cm⁻¹ longer than that of 13- cis -bR; the molar extinction coefficient (ε) is about 74000 M ⁻¹ cm⁻¹, larger than that of 13- cis -bR (52000 M ⁻¹ cm⁻¹).
On the warming the sample containing batho-bR¹³ formed by irradiating 13- cis -bR or bR^D at — 190°C, we could not detect other intermediates such as the lumi- or meta-intermediates seen in trans-bR system.     

BIOPHYSICAL AND BIOCHEMICAL ASPECTS OF PHENGODID (RAILROAD-WORM) BIOLUMINESCENCE

In studies of the bioluminescence of 11 species of phengodid collected in central and southeast Brazil, we have found that: (1) their lateral lanterns emit light in the yellow-green region (λ_max= 540–580 nm) and the head lantern color is shifted to the red region ( λ_max= 565–620 nm), (2) the luciferins of both types of lanterns are identical to that of lampyrids and elaterids and (3) the luciferase physicochemical properties are also similar to those of lampyrids and elaterids (optimum pH ca 8.1; K_m(ATP) = 260–370 μM , Kμ(luciferin) = 170–400 μM; molecular weight ca 60 kDa; apparent activation energy of in vitro bioluminescence ca 58 kJ/mol). Thus the bioluminescence system of phengodids appears to be essentially the same as that of lampyrids and elaterids. The different bioluminescence colors of the lanterns of Phrixothrix species (λ_head= 600–620 nm; λ_lateral= 535–565 nm) and other phengodid species are probably elicited by the presence of luciferase isoenzymes, as occurs in the case of elaterid prothoracic and abdominal lanterns.     

RECONSTITUTION OF SQUID RHODOPSIN IN RHABDOMAL MEMBRANES

Abstract— Squid opsin which is capable of combining with 11- cis or 9- cis retinal to reconstitute photo-pigment has been prepared by irradiation of rhabdomal membranes with orange light (> 530 nm) in the presence of 0.2 M hydroxylamine. When the irradiation is carried out either at concentrations of hydroxylamine higher than 0.2 M or with light of wavelength shorter than 530 nm, rhodopsin in the membranes is bleached quickly, but the ability of the resultant opsin to form rhodopsin is greatly reduced.
The optimum pH for rhodopsin regeneration in rhabdomal membranes was found to be between 6.5 and 8.5. The rate of regeneration of rhodopsin increases with raising temperature, and at about 20°C it is almost the same as that of isorhodopsin. Even after solubilization in digitonin solution, opsin still preserves the ability to reform rhodopsin.
All- trans retinal can be incorporated into retinochrome-bearing membranes, in which it is isomerized into 11- cis isomer by the photoisomerase activity of retinochrome. Rhabdomal membranes retaining active opsin can take up 11- cis retinal from retinochrome membranes so as to synthesize rhodopsin.     

LASER PHOTOLYSIS OF RETINAL AND ITS PROTONATED AND UNPROTONATED n-BUTYLAMINE SCHIFF BASE

Abstract —The pulsed ruby laser (347 nm) flash photolysis technique has been used to measure the triplet-triplet absorption spectra and triplet lifetimes of trans -retinal, N-frans -retinylidene- n -butylamine (NRBA), and protonated NRBA (NRBAH⁺) at room temperature. In methylcyclohexane solution, the triplet lifetimes are in the range 10–20 μs and decrease in the order NRBAH⁺ > NRBA > trans -retinal. Intersy stem-crossing efficiencies (φ_ISC) were determined by a comparison technique using anthracene and 1,2-benzanthracene as reference compounds. For trans -retinal, φ_ISC is 0–50 pM 0–05 in methylcyclohexane and 0–08 in methanol, which confirms that earlier values of 0–11 and 0–017 in these solvents are in error. For NRBA and NRBAH⁺ in methylcyclohexane, Φ_ISC values are 0008 and < 0–001, respectively. Evidence is presented for a significant solvent effect in the isomerization of retinal via the triplet state, and that cis φ trans isomerization occurs from the triplet state of NRBAH⁺. The relation between the intersystem-crossing properties of model compounds and the photochemistry of rhodopsin is discussed.     

FLUORESCENCE DETECTION OF AN 8-METHOXYPSORALEN METABOLITE IN HUMAN INTERSTITIAL FLUID

Abstract— A fluorescent method has been used to study the suction blister fluid of human volunteers collected after 8-methoxypsoralen (8-MOP) oral intake. A fluorescent chromophore with spectral characteristics (Λ_max= 390 nm, Λ_max=470nm) distinct from 8-MOP has been detected. Our results suggest the existence of a metabolite form of 8-MOP within the patients's skin prior to any UV irradiation. This form might result in the opening of the4–5' double bond of the 8-MOP molecule.     

TRANSIENT PHENOMENA IN THE PULSE RADIOLYSIS OF RETINYL POLYENES—7. RADICAL ANIONS OF VITAMIN A AND ITS DERIVATIVES

Abstract— Upon e^--pulse irradiation in nonprotic solvents, all- trans retinol (ROH) and retinylmethyl ether (ROMe) form transient species (τ= 0.5–7μs, λ_max=575–590 nm) identifiable as radical anions. Similar species are also formed upon laser pulse photoexcitation of these retinyl derivatives in the presence of N,N-dimethylaniline in acetonitrile. In contrast, electron transfer or attachment to all- trans retinyl acetate (ROAc) and palmitate (ROPa) results in 'instantaneous' loss of carboxylate anions from electron adducts giving the retinylmethyl radical (R-, λ_max= 395 nm, τ_k > 100 μ,s); the radical anions in these cases are too short-lived to be detected by nanosecond pulse radiolysis. The lifetimes of radical anions of ROH and ROMe are very sensitive to water and alcohols (e.g. k_q = 10⁷ M ^-1 s^-1 with methanol as quencher for ROH^- in tetrahydrofuran). Based on these findings, the spectral dissimilarity of the one-electron reduction products from ROH and ROAc in alcohols and aqueous micelles becomes explainable in terms of fast formation of protonated radical anions (RH(OH), τ_1/2, > 100 μs, λ_max=370–375 nm) in the case of ROH and of retinylmethyl radical via loss of AcO^- from radical anion in the case of ROAc. In tetrahydrofuran, the complexation of ROH^- with cations such as Na⁺ and Bu₄N⁺ affects the relative importance of its major decay modes, namely, protonation and dehydroxylation, the latter process being significantly enhanced by the presence of Na⁺.     

BIOLUMINESCENCE OF THE BRITTLE STAR OPHIOPSILA CALIFORNICA

The light-emitting principle of the brittle star Ophiopsila californica has been isolated and purified. It was found to be a green-fluorescent photoprotein (molecular weight 45000) which emits green light (λ_max 500 nm) when H₂O₂ is added, independently of the presence or absence of O₂. The green fluorescence (emission maximum 500 nm, excitation maximum 440 nm) spectrally coincided with the H₂O₂-triggered luminescence, indicating that the green fluorescent chromophore is the light-emitter of the photoprotein luminescence.     

THE FORMATION OF TWO FORMS OF BATHORHODOPSIN AND THEIR OPTICAL PROPERTIES

Abstract— Using two kinds of rhodopsin preparations (digitonin extract and rod outer segments suspension), we measured changes in absorption spectra during the conversion of rhodopsin or isorhodopsin to a photosteady state mixture composed of rhodopsin, isorhodopsin and bathorhodopsin by irradiation with blue light (437 nm) at 77 K and during the reversion of bathorhodopsin to a mixture of rhodopsin and isorhodopsin by irradiation with red light (> 650 nm) at 77 K. The reaction kinetics could be expressed with only one exponential in the former case and with two exponentials in the latter case. These data suggest that both rhodopsin and isorhodopsin are composed of a single molecular species, while bathorhodopsin is composed of two molecular species, designated as bathorhodopsin₁ and bathorhodopsin₂. The absorption spectra of these bathorhodopsin were calculated by two different methods (kinetic method and warming-cooling method). The former was based on the kinetics of the conversion of two forms of bathorhodopsin by irradiation with the red light. The spectra obtained by this method were consistent with those obtained by the warming-cooling method. Bathorhodopsin₁ and bathorhodopsin₂ have Λ_max at 555 and 538 nm, respectively. The two forms of bathorhodopsin are interconvertible in the light, but not in the dark. Thus, we suggest that a rhodopsin molecule in the excited state relaxes to either bathorhodopsin₁ or bathorhodopsin₂ through one of the two parallel pathways.     

CHROMOPHORE OF A LONG-LIVED PHOTOPRODUCT FORMED WITH METARHODOPSIN III IN THE ISOLATED FROG RETINA

Abstract Long-lived photoproducts of frog rhodopsin in isolated retina and digitonin solution have been investigated by spectrophotometry and their chromophores have been analyzed by high-pressure liquid chromatography (HPLC). By irradiation (> 560 nm) at 3°C and pH 8.6, a product analogous to metarhodopsin III (MIII) is formed, whose absorption maximum is at about 450 nm. This product decays more slowly than MIII does. The results of HPLC analysis indicate that the chromophore of this photoproduct is 7- cis retinal and that of MIII is all-trans retinal. The product possessing 7- cis retinal is called 7- cis photoproduct. The amount of 7- cis isomer in rhodopsin solution irradiated at various temperatures between 15°C and –82°C, has been determined. The results suggest that the 7- cis photoproduct can be formed by the photoconversion of lumirhodopsin and metarhodopsin I.     

SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN-FLUORESCENT PROTEINS

Abstract— Spectral properties of guanidine-denaturated and pronase-digested green-fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6 M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine -HCl. Pronase digestion of the heat-denaturated GFP's generated a methanol-soluble blue-fluorescent peptide with identical fluorescence emission spectra (λ_max= 430 nm, uncorrected; φ_f1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.     

THEORY OF THE OPTICAL ABSORPTION OF LIGHT-ADAPTED BACTERIORHODOPSIN AND ITS ACIDIFIED FORMS

Abstract— We assume a model for bacteriorhodopsin chromophore such that the protonated retinal Schiff-base (PRSB) interacts with two anions in the case of light-adapted bacteriorhodopsin (bR^L), while it does with one anion in the case of the acidified form of bacteriorhodopsin (bR^acid₆₀₀). On the basis of this model, the π-electronic states of all- trans -PRSB are calculated according to our LCAO-ASMO-SCF-CI method, the anions being approximated by negative point-charges in the plane of PRSB π-system. A possible distribution of the negative point-charges around PRSB is proposed for the chromophores of bR^L, bR^acid₆₀₀, and the two irradiated forms of bR^acid₆₀₀ (the one at 3°C containing 9- cis -PRSB, and the other at — 72°C all- trans -PRSB). It is shown that the wavelength λ_max of absorption maximum observed for each form of bacteriorhodopsin can be explained reasonably well by the suggested charge distribution. Furthermore, a model for the structure of the active site of bR^L is proposed, considering that two COO⁻ groups form the anions that interact with PRSB. The calculated optical absorption of all- trans -PRSB at such a site is shown to be consistent with the observed absorption spectrum of bR^L.     

ABSORPTION SPECTRA OF TCA-DENATURED RHODOPSIN AND OF A SCHIFF BASE COMPOUND OF RETINAL

Abstract— When TCA-denatured rhodopsin was frozen in liquid nitrogen, Λ_max was markedly shifted to longer wavelengths as the concentration of TCA increased. After TCA denaturation, species specific absorption disappeared and the absorption maxima of the squid pigments became identical with those of corresponding pigments of octopus.
In solutions at 5° the bathochromic shift of Λ_max of TCA denatured rhodopsin was observed at higher concentrations of TCA than in the frozen state. Λ_max of N-retinylidene-butylamine (NRB) was also displaced towards longer wavelengths with increasing concentrations of TCA. This bathochromic shift was enhanced by freezing. The mode of the bathochromic shift of Λ_max provoked by TCA was very similar both in the cases of denatured rhodopsin and of NRB. The absorption spectrum of NRB was identical in shape with that of TCA-denatured rhodopsin, as the half-band widths of both materials were about 5500 cm^-1 in the liquid state and 5000 cm^-1 in the frozen state. Λ_max of retinal and NRB were red shifted in polar and polarizable solvents.
It was concluded that the strong acidity and the relatively large polarizability of TCA are responsible for the bathochromic shift of Λ_max of the Schiff base in TCA-denatured rhodopsin.     

LIGHT INDUCED ISOMERISATION, AT ACIDIC pH, INITIATES HYDROLYSIS OF BACTERIORHODOPSIN TO BACTERIO-OPSIN AND 9-CIS-RETINAL

Abstract— Bacteriorhodopsin (BR) from the purple membrane of Haiobacterium halobium contains covalently bound retinal in the 13- cis and all- trans configurations. Several forms of bacteriorhodopsin are known, with different absorption maxima which are designated as BRλ_max (nm). At acidic pH, BR605 is formed from BR560. The following sequence of reactions was found, which is initiated by irradiation of BR605 with red light:

An all- trans /13- cis to 9- cis isomerisation occurs in the light induced reaction BR605 ∼ BR500. BR500 seems to contain covalently bound retinal, whereas BR390 contains free retinal. By irradiation with light, BR500, BR450 and BR390 can be reconverted to BR560.     

QUANTUM EFFICIENCY AND PHOTOSENSITIVITY OF THE RHODOPSIN ⇌ METARHODOPSIN CONVERSION IN CRAYFISH PHOTORECEPTORS

Photosensitivity (K_λ) of a visual pigment is the product of the molecular absorption coefficient (α_λ) and the quantum efficiency for photoconversion (γ). Among the invertebrates, many visual pigments are stable not only in the rhodopsin (R) conformation but also as the photoproduct, metarhodopsin (M), We here employ a method for determining the photosensitivities of the two stable pigments of a rhodopsin-metarhodopsin pair, using kinetic analysis of fluorescence from metarhodopsin combined with measurements of spectral absorption made before and after saturation at the isosbestic wavelength of the pigment pair. A curve fitting technique, in which a theoretical function is scaled for best fit to the measured absorption spectrum of the photosteady-state mixture, yields values for the photosensitivity of rhodopsin at λ._max, the ratio of quantum efficiencies for rhodopsin—metarhodopsin interconversion, and the fractional composition of the steady-state mixture. With knowledge of the molecular extinction coefficient, the absolute values of quantum efficiency can be calculated. For crayfish ( Orconectes, Procambarus ) rhodopsin, measured in isolated rhabdoms, K_max= 1.05 x 10^-16 cm² at 535 nm with >7λR→M0.69. These values are similar to the photosensitivity and quantum efficiency of bleaching of vertebrate rhodopsins in digitonin solution (Dartnall, 1972). For the metarhodopsin, K_max= 1.02 x 10^-16 cm² at 510 nm, and λ_M-R= 0.49.     

PHOTOCHROMISM OF 2,6-DINITRODIPHENYL ETHERS ENHANCED IN THE PRESENCE OF A HOST BIOMOLECULE*

Abstract— The photochromic behavior of 2,6-dinitrodiphenyl ethers is described. In the course of a study for the photoaffinity labeling of the binding site for thyroxine on human prealbumin, it was found that the thyroxine analog 3-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-dinitrophenyl]propionic acid (1), on irradiation with >350nm light in the presence of prealbumin, gave a pink species (λ_max 508 nm). In the absence of prealbumin, no color formation was observed. Irradiation of 3-[4-(4-hydroxyphenoxy-3,5-dinitrophenyl]propionic acid (2) and 3-(4-phenoxy-3,5-dinitrophenyl)propionic acid (3), analogs of 1, in acetonitrile, aqueous bicarbonate, or ethanol gave a colored species (λ_max 500–520 nm depending on the solvent) in the absence of a host molecule. In the presence of β-cyclodextrin, which may be considered a model for a binding site on a protein, photocoloration occurred at a faster rate than in the absence of ß-cyclodextrin. For both 2 and 3 , the colored species underwent thermal back reaction to the starting compound. The activation energy for the thermal back reaction was estimated to be about 40 kj/mol.     

PHOTOREACTION OF THE ACIDIFIED FORM OF BACTERIORHODOPSIN AND ITS 9-CIS DERIVATIVE IN PURPLE MEMBRANE AT LOW TEMPERATURES

Abstract— The photoreaction of the acidified form of bacteriorhodopsin and its 9-cis derivative was studied by low temperature spectroscopy.
A short exposure of the acidified form of bacteriorhodopsin, which was prepared by adding 2 m M HC1 to purple membrane suspension in 67% glycerol at 0°C, to red light at – 72°C resulted in the blue-shift of the spectrum. The feature of the shift was very similar to that accompanied by the formation of stable 9- cis acidified form of bacteriorhodopsin at 0°C, but only 13- cis - and all- trans -retinals were found in the extract from this product. No blue-shifted product was found on irradiation at – 190°C.
Irradiation of the 9- cis form of acidified bacteriorhodopsin at -72°C with blue light caused the isomerization of its 9- cis -retinylidene chromophore to 13- cis and all- trans forms without a significant spectral change. It became greater only after the sample was warmed above – 24°C. These results indicate the presence of the light-induced product which has trans configuration on the 9-10 double bond and exhibits the 9- cis type spectrum.     

QUALITATIVE STUDIES OF THE LOW TEMPERATURE PHOTOCHEMISTRY OF RHODOPSIN AND RELATED PIGMENTS

Abstract— Rhodopsin, the isomeric pigments formed from 9- cis - and 9, 13- dicis -retinal, and the synthetic pigments formed from 9- cis - and 11- cis -14-methylretinal were irradiated with 490 nm light at -196C. Absorption spectral changes indicate that a distinguishable bathorhodopsin type intermediate may be formed for each pigment. The bathorhodopsin intermediates of the 9- cis pigments have band maxima hypsochromically shifted by4–5 nm compared to their corresponding rhodopsins. The bathorhodopsin type intermediate formed upon irradiation of 9, 13- dicis -rhodopsin has an absorption that maximizes 6 nm shorter than that of rhodopsin. Band maxima of the bathorhodopsin intermediates of the 14-methylrhodopsins are bathochromatically shifted ca. 8 nm compared to their corresponding rhodopsins.     

PHYTOCHROME INTERMEDIATES IN VIVO-I. EFFECTS OF TEMPERATURE, LIGHT INTENSITY, WAVELENGTH AND OXYGEN ON INTERMEDIATE ACCUMULATION

Abstract— Accumulation of weakly absorbing phytochrome intermediates has been demonstrated in Pisum epicotyl tissue under conditions of pigment cycling using a quasi-continuous measuring spectrophotometer. An action spectrum shows 690–700 nm to be the most efficient wavelength range in this process. Difference spectra for the decay of intermediates maintained by 690 nm light show that, if the experiment is done at 0°C, only P_fr is formed. At – 11°C, intermediates decaying to P_r can also be observed. At – 20°C, P_r is produced as well as a pigment with peak absorption at 710nm. Kinetic analysis of intermediate decay at – 11°C reveals that at least two intermediates are maintained by 690 nm light. The level of intermediate maintained by incandescent light at 0°C was 25% higher in air than in nitrogen.