PHOTOCHEMISTRY OF VISUAL PURPLE : II. THE EFFECT OF TEMPERATURE ON THE BLEACHING OF VISUAL PURPLE BY LIGHT.

The temperature coefficient of the bleaching of visual purple by light is 1.00 over a range of 30 degrees. This indicates that the monomolecular course of the reaction represents a real chemical process, as opposed to a possible diffusion process, and that the reaction is probably simple in nature.     

PHOTOCHEMISTRY OF VISUAL PURPLE : III. THE RELATION BETWEEN THE INTENSITY OF LIGHT AND THE RATE OF BLEACHING OF VISUAL PURPLE.

It is shown that the velocity of bleaching of visual purple by light, under comparable conditions of concentration, volume, and surface exposed, is directly proportional to the intensity.     

ANOMALIES IN THE ABSORPTION SPECTRUM AND BLEACHING KINETICS OF VISUAL PURPLE

Visual purple from winter frogs shows an intermediate yellow color during bleaching by light; summer extractions do not. This seasonal effect can be duplicated by variations in the hydrogen ion concentration and in the temperature of the solutions. Increasing the pH approximates the summer condition, while decreasing the pH approximates the winter condition. Temperature has no effect on the bleaching of alkaline solutions but greatly influences acid solutions. At low temperatures the bleaching of add solutions resembles the winter condition, while at higher temperatures it resembles the summer condition. A photic decomposition product of frog retinal extractions is an acid-base indicator: it is yellow in acid and colorless in alkaline solution. Its color is not dependent upon light. The hydrogen ion concentration of visual purple solutions does not change under illumination, nor is there a difference in the pH of summer and winter extractions. Bile salt extractions of visual purple are usually slightly acid. The conflicting results of past workers regarding the appearance of "visual yellow" may be due to seasonal variation with its differences in temperature, or to the presence of base in the extractions. It is also possible that vitamin A may be a factor in the seasonal variation. The photic decomposition of visual purple in bile salts solution, extracted from summer frogs, follows the kinetics of a first order reaction. Visual purple from winter frogs does not conform to first order kinetics. Photic decomposition of alkaline, winter visual purple extractions also follows a first order equation. Acid, winter extractions appear to conform to a second order equation, but this is probably an artefact due to interference by the intermediate yellow.     

REGENERATION OF VISUAL PURPLE IN SOLUTION

1. Measurements of visual purple regeneration in solution have been made by a procedure which minimized distortion of the results by other color changes so that density changes caused by the regenerating substance alone are obtained. 2. Bleaching a visual purple solution with blue and violet light causes a greater subsequent regeneration than does an equivalent bleaching with light which lacks blue and violet. This is due to a photosensitive substance which has a gradually increasing effective absorption toward the shorter wavelengths. It is uncertain whether this substance is a product of visual purple bleaching or is present in the solution before illumination. 3. The regeneration of visual purple measured at 560 mµ is maximal at about pH 6.7 and decreases markedly at more acid and more alkaline pH''s. 4. The absorption spectrum of the regenerating material shows only a concentration change during the course of regeneration, but has a higher absorption at the shorter wavelengths than has visual purple before illumination. 5. Visual purple extractions made at various temperatures show no significant difference in per cent of regeneration. 6. The kinetics of regeneration is usually that of a first order process. Successive regenerations in the same solution have the same velocity constant but form smaller total amounts of regenerated substance. 7. In vivo, the frog retina shows no additional oxygen consumption while visual purple is regenerating.     

THE REGENERATION OF VISUAL PURPLE IN THE LIVING ANIMAL

1. The accumulation of visual purple in the retina after bleaching by light has been studied in the intact eye of the frog. The data show that duration and intensity of light adaptation, which influence the course of human dark adaptation as measured in terms of visual threshold, have a similar influence on the course of visual purple regeneration. 2. At 25°C. frogs which have been light adapted to 1700 millilamberts and then placed in the dark, show an increase in visual purple concentration which begins immediately and continues for 70 minutes until a maximum concentration is attained. The increase, although beginning at once, is slow at first, then proceeds rapidly, and finally slows up towards the end. Frogs which have been adapted to 9500 millilamberts show essentially the same phenomenon except that the initial slow period is strongly delayed so that almost no visual purple is formed in the first 10 minutes. 3. At 15°C. the initial delay in visual purple regeneration occurs following light adaptation to both 1700 and 9500 millilamberts. The delay is about 10 minutes and is slightly longer following the higher light adaptation. 4. The entire course of visual purple accumulation in the dark takes longer at the lower temperature than at the higher. The temperature coefficient for 10°C. is about 1.8. 5. In contrast to the behavior of the isolated retina which has small amounts of vitamin A and large amounts of retinene immediately after exposure to light, the intact eye has large amounts of vitamin A and little retinene after exposure to light for 10 minutes. In the intact eye during dark adaptation, the amount of vitamin A decreases markedly while retinene decreases only slightly in amount. If retinene is formed in the intact eye, the change from retinene to vitamin A must therefore occur rapidly in contrast to the slow change in the isolated retina. 6. The course of visual purple regeneration may be described by the equation for a first order autocatalyzed reaction. This supposes that the regeneration of visual purple is catalyzed by visual purple itself and accounts for the sigmoid shape of the data.     

THE ABSORPTION SPECTRUM OF VISUAL PURPLE

The absorption spectra of visual purple solutions extracted by various means were measured with a sensitive photoelectric spectrophotometer and compared with the classical visual purple absorption spectrum. Hardening the retinas in alum before extraction yielded visual purple solutions of much higher light transmission in the blue and violet, probably because of the removal of light-dispersing substances. Re-extraction indicated that visual purple is more soluble in the extractive than are the other colored retinal components. However, the concentration of the extractive did not affect the color purity of the extraction but did influence the keeping power. This suggests a chemical combination between the extractive and visual purple. The pH of the extractive affected the color purity of the resulting solution. Over the pH range from 5.5 to 10.0, the visual purple color purity was greatest at the low pH. Temperature during extraction was also effective, the color purity being greater the higher the temperature, up to 40°C. Drying and subsequent re-dissolving of visual purple solutions extracted with digitalin freed the solution of some protein impurities and increased its keeping power. Dialysis against distilled water seemed to precipitate visual purple from solution irreversibly. None of the treatments described improved the symmetry of the unbleached visual purple absorption spectrum sufficiently for it to resemble the classical absorption spectrum. Therefore it is very likely that the classical absorption spectrum is that of the light-sensitive group only and that the absorption spectra of our purest unbleached visual purple solutions represent the molecule as a whole.     

THE VISIBILITY OF MONOCHROMATIC RADIATION AND THE ABSORPTION SPECTRUM OF VISUAL PURPLE

1. After a consideration of the existing data and of the sources of error involved, an arrangement of apparatus, free from these errors, is described for measuring the relative energy necessary in different portions of the spectrum in order to produce a colorless sensation in the eye. 2. Following certain reasoning, it is shown that the reciprocal of this relative energy at any wave-length is proportional to the absorption coefficient of a sensitive substance in the eye. The absorption spectrum of this substance is then mapped out. 3. The curve representing the visibility of the spectrum at very low intensities has exactly the same shape as that for the visibility at high intensities involving color vision. The only difference between them is their position in the spectrum, that at high intensities being 48 µµ farther toward the red. 4. The possibility is considered that the sensitive substances responsible for the two visibility curves are identical, and reasons are developed for the failure to demonstrate optically the presence of a colored substance in the cones. The shift of the high intensity visibility curve toward the red is explained in terms of Kundt''s rule for the progressive shift of the absorption maximum of a substance in solvents of increasing refractive index and density. 5. Assuming Kundt''s rule, it is deduced that the absorption spectrum of visual purple as measured directly in water solution should not coincide with its position in the rods, because of the greater density and refractive index of the rods. It is then shown that, measured by the position of the visibility curve at low intensities, this shift toward the red actually occurs, and is about 7 or 8 µµ in extent. Examination of the older data consistently confirms this difference of position between the curves representing visibility at low intensities and those representing the absorption spectrum of visual purple in water solution. 6. It is therefore held as a possible hypothesis, capable of direct, experimental verification, that the same substance—visual purple—whose absorption maximum in water solution is at 503 µµ, is dissolved in the rods where its absorption maximum is at 511 µµ, and in the cones where its maximum is at 554 µµ (or at 540 µµ, if macular absorption is taken into account, as indeed it must be).     

紫膜与溶剂的相互作用

本文研究了溶剂正己烷,正十六烷,甲苯和二甲基甲酰胺(DMF dimethyl formamide)与紫膜的相互作用.吸收光谱,园二色谱和紫膜光循环中间产物M412的动力学过程的测量表明,在不同条件下,溶剂与紫膜能相互作用而影响到紫膜的光谱特性和光化学循环动力学过程.结果说明,在制作紫膜LB膜时,正己烷和正十六烷是合适的,使用二甲基甲酰胺时必须防止强光照射,甲苯则不能采用.     

CAROTENOIDS AND THE VISUAL CYCLE

1. Carotenoids have been identified and their quantities measured in the eyes of several frog species. The combined pigment epithelium and choroid layer of an R. pipiens or esculenta eye contain about 1γ of xanthophyll and about 4γ of vitamin A. During light adaptation the xanthophyll content falls 10 to 20 per cent. 2. Light adapted retinas contain about 0.2–0.3 γ of vitamin A alone. 3. Dark adapted retinas contain only a trace of vitamin A. The destruction of their visual purple with chloroform liberates a hitherto undescribed carotenoid, retinene. The bleaching of visual purple to visual yellow by light also liberates retinene. Free retinene is removed from the isolated retina by two thermal processes: reversion to visual purple and decomposition to colorless products, including vitamin A. This is the source of the vitamin A of the light adapted retina. 4. Isolated retinas which have been bleached and allowed to fade completely contain several times as much vitamin A as retinas from light adapted animals. The visual purple system therefore expends vitamin A and is dependent upon the diet for its replacement. 5. Visual purple behaves as a conjugated protein in which retinene is the prosthetic group. 6. Vitamin A is the precursor of visual purple as well as the product of its decomposition. The visual processes therefore constitute a cycle.     

血卟啉衍生物（HPD）对紫膜菌紫质（bR）的光敏化作用

研究了血卟啉衍生物（ＨＰＤ）对嗜盐菌紫膜上蛋白质菌紫质（ｂＲ）的光敏化作用,结果表明,ＨＰＤ与紫膜结合并不影响ｂＲ的光学性质及活性;但经光照射、ＨＰＤ光敏反应后,ｂＲ丧失光循环活性。进一步的探测显示ｂＲ中的视黄醛色素团及色氨酸均在光敏反应中受损,反映了除视黄醛色素团有可能直接受损外,深埋于折叠蛋白内部的部分色氨酸残基。亦可能在ＨＰＤ光敏化过程中被损伤。实验证明,单线态氧（＾１Ｏ２）的作用是ＨＰＤ光     

酰化对紫膜结构的影响

在很宽波长和ＰＨ范围内用紫外及可见区光吸收、圆二色及共振拉曼测定了酰化诱导的紫膜溶液的光谱变化。结构表明：酰化引起的表面电位的改变诱导了蛋白局部构象的改变。这种改变在很大程度上离域到整个蛋白分子中而影响了视黄醛结合位点的性质及生色团的结构,从而导致可观察的光谱变化。与未酰化紫膜比较,紫膜对ＰＨ的稳定性减小了。     

紫膜LB膜的结构特性研究

研究了紫膜LB膜中的紫膜碎片的结构特性。扫描电子显微镜观察表明,紫膜LB膜中单个紫膜碎片的直径大约为0.3微米。表面轮廓测量仪(简称台阶仪)观察到紫膜LB膜中的紫膜碎片的厚度为40—50。在不同的表面压和不同紫膜含量时测量了紫膜碎片在紫膜LB膜中的形态学分布,当表面压为30mN/m或紫膜与大豆磷脂的重量比大于20:1时,紫膜碎片容易重叠或凝聚。     

生物素结合对紫膜结构和功能的影响

为了提高细菌视紫红质（ＢＲ）的可组装怀,使之使用于生物传感器等生物器件,利用生物素对紫膜进行修饰,使之可以被亲和素识别,从而可以定向的固定于固体支撑物表面。实验结果表明：生物素可以修饰紫膜表面的赖氨酸,修饰的程序依修饰的时间不同而有所不同,但即使被修饰２４小时的紫膜,其表面的赖氨酸仍然没有被完全修饰。同时,生物素的修饰不会影响紫膜的结构和功能,但是不同的结合位点对Ｍ４１２的衰减会产生不同的影响。这     

表面压与紫膜含量对紫膜LB膜质量的影响

本文报导了用表面轮廓测量仪测量了不同表面压和不同紫膜含量下制备的紫膜LB膜中紫膜碎片的厚度。实验结果表明:单个紫膜碎片在紫脂LB膜中的厚度为50左右,相当数量的紫膜碎片之间有重叠。当表面压为30mN/m或紫膜碎片与大豆磷脂之重量比为20:1时,紫膜碎片容易进入到水相或碎片之间相互重叠变得更加严重。     

中性红再生紫膜的光化学性质

研究了中性红再生紫膜从先适应状态到暗适应状态的反应及再生紫膜中中性红的光吸收变化。实验结果说明紫膜上的金属离子结合位点可能深入膜内的质子通道,与构成质子通道的一些重要氨基酸残基相互作用。紫膜经去离子化处理变成蓝膜后,带有正电荷的质子化中性红也可以占据此金属离子结合位点,使蓝膜再生为紫膜。但金属离子与结合位点具有更强的亲和力,使蓝膜再生为紫膜的能力比质子化中性红强。     

紫膜的紫外荧光及分子内的能量转移

以可见光为作用光照射天然紫膜,紫膜蛋白被280nm紫外光激发所发射的荧光强度比对照略有降低.比较天然紫膜、漂白紫膜与菌蛋白三者的紫外荧光强度,前两者无显著变化,但菌蛋白的荧光强度比天然紫膜的荧光强度大2-3倍,表明生色团对蛋白质荧光可能有猝灭作用.用280nm波长光照射紫膜的暗适应形式,可使其转变成光适应形式.若有羟胺存在,以紫外光照射也可使紫膜漂白.光漂白的作用光谱,其紫外部分与紫膜蛋白部分的吸收光谱重合得很好.上述实验证明紫膜蛋白部分吸收的能量可以转移到生色团上,即紫膜存在分子内的能量转移     

嗜菌紫膜表面电位和质子泵效率间的关系

本文研究了中性红与紫膜结合,并用直接滴定法和测定结合量法,求得了结合于膜上之中性红本征PK和在不同离子强度时的表观PKa值.从它们的差值中计算得到紫膜表面电位.并得不同盐浓度的表面电位和质子泵效率作了比较.结果说明.阳离子对质子泵效率的影响不能完全用表面电位的变化来说明.这一结果暗示了一定的阳离子对于紫膜质子泵功能的完成有着特殊作用.     

令箭荷花紫色素稳定性的研究

研究了酸度、热、光、淀粉、糖、抗坏血酸、苯甲酸钠、金属离子对令箭荷花紫色素稳定性的影响。结果表明：该色素在酸性、中性稳定;光、热对色素有一定的降解作用;淀粉、糖、抗坏血酸、Na＋、Zn2＋对色素无不良影响;苯甲酸钠、Fe3＋、Cu2＋对色素有一定影响。     

THE R?LE OF ORGANIC SUBSTRATES IN PHOTOSYNTHESIS OF PURPLE BACTERIA

A representative of the photosynthetic non-sulfur purple bacteria (Athiorhodaceae) capable of using simple alcohols has been isolated in pure culture. By means of quantitative analysis of cultures at different stages of development it has been shown that this organism converts isopropanol quantitatively into acetone, simultaneously reducing CO₂ in the light. The data can be represented by the equation 2 CH₃CHOHCH₃ + CO₂ → 2 CH₃COCH₃ + (CH₂O) + H₂O. Manometric experiments with suspensions of resting cells have fully corroborated the results obtained with growing cultures. The experiments have conclusively proved that an organic substrate may fulfill exclusively the function of hydrogen donor for the photochemical CO₂-reduction in purple bacteria photosynthesis.     

山莨菪碱对紫膜中菌紫质结构的影响及其与膜脂的关系

本文用吸收光谱和可见圆二色谱研究了不同浓度的山莨菪碱对紫膜中菌紫质结构的影响,并设计了用不同浓度的去垢剂Triton X-100作为脂环境的扰动剂,研究山莨菪碱对菌紫质的影响与膜脂关系的实验.结果表明山莨菪碱不仅影响菌紫质分子本身的构象变化而且扰动了菌紫质分子之间的激子偶联作用.通过吸收差光谱技术表明山莨菪碱对菌紫质结构的影响与膜脂密切相关并指出紫膜中菌紫质的三体结构对膜功能的贡献是不容忽视的.