Nonbleachable Pigment Research Articles

Solubility of Quercetin in Wines

Quercetin solubility at 18 °C and 0 °C was determined in an hydroalcoholic buffer solution with pH 3.20 and in four Italian wines to study the formation of quercetin precipitate mechanism in wines. The wines selected were Barbera 2018 for the typically high content in bisulfite bleachable pigments, red Cirò 2014, for its typically high content of flavonoids, Sangiovese 2014, for the presence of quercetin deposits in bottle, and white Cirò 2018, for the absence of red pigments. All the samples were spiked with 30 mg/L quercetin. The amount of quercetin solubilized at 18 °C and 0 °C in the hydroalcoholic buffer was much lower than in wines, while that solubilised in Barbera was much higher compared to red Cirò, Sangiovese and white Cirò. Solubilised quercetin was lower in all wine samples stored at 0 °C than in those at 18 °C. The pigment composition of the three red wines examined suggested that the over-solubility of quercetin could be due to the formation of soluble co-pigmentation complexes between quercetin and monomer anthocyanins and/or bisulphate-bleachable flavanol-anthocyanin pigments. A positive correlation between quercetin solubility and bleachable pigment was noted: the richer the wine in bleachable pigments, the higher the solubility of quercetin. Quercetin haze formation appeared due to the release of quercetin from co-pigmentation complexes during wine maturation and storage ,as its counterpart, anthocyanins, form non-bleachable pigments or are degraded in hydrolytic or oxidation reactions. Quercetin in aged red wines seemed to reach a content similar to that of white wine spiked with quercetin. Nevertheless, the quercetin content of aged red wines in which a quercetin haze has been found could be lower than that when added white wine, due to its degradation probably being induced by oxidation reactions. Finally, the solubilized quercetin content in white Cirò, which was higher than the hydroalcoholic buffer solution, suggested that there may be different substances in wines than pigments that prevent the growth of quercetin crystals. However, their nature was not determined in this study.

Anthocyanins from purple passion fruit (Passiflora edulia Sims) rind-An innovative source for nonbleachable pigment production.

Passion fruit rind is a waste product from the beverage industry that is rich in anthocyanins that can be potentially applied as a natural colorant. However, the inherent instability of anthocyanins causes rapid discoloration. In this study, the cyanidin-3-glucoside (C-3-G) in passion fruit rind was extracted using 50% ethanol and converted into nonbleachable pigments by reaction with Oolong tea extracts and acetaldehyde. Reactions over 70 days formed high concentrations of stable nonbleachable pigments (3.07-6.68 absorbance unit [A.U.], in total) such as pyranoanthocyanins, as well as oligomeric and polymeric pigments with ethyl-linked bridges. In C-3-G and acetaldehyde reaction, positive relations were found among acetaldehyde concentration, color density, and nonbleachable pigment concentrations. As for reactions with C-3-G and Oolong tea extract combined with acetaldehyde, greater color density and greater concentrations of nonbleachable pigments (10.80-12.34, 4.25-4.40 A.U., respectively) were formed compared with acetaldehyde alone. In addition, the antioxidant capabilities of the extracts were enhanced after reaction with Oolong tea extracts. The results of this study show a useful method to enhance the stability of anthocyanins from passion fruit rind and also provide greater economic value to this waste product. PRACTICAL APPLICATION: Ripened passion fruits contain a high concentration of anthocyanins in their rind. These anthocyanins can be optimally extracted by ultrasonic assisted solvent extraction to provide stable pigments by inducing acetaldehyde (a volatile compound often found in foods and beverages) into the anthocyanins. These stable pigments have a greater reddish hue in solution than the anthocyanin extracted from the rind and are more stable over a greater pH range. In addition, these stable pigments can be potentially used as colorant throughout the food and cosmetic industry to provide high economical values.

Precipitation of Tannin-Anthocyanin Derivatives in Wine is Influenced by Acetaldehyde Concentration and Tannin Molecular Mass with Implications for the Development of Nonbleachable Pigments.

This study aimed to assess the effect of tannin molecular mass on nonbleachable pigment formation and subsequent stability under wine-like conditions. Tannin fractions of a defined molecular mass range were prepared from grape skins and seeds and reacted with malvidin-3-glucoside for 120 days in three media types: chemically defined wine media with or without acetaldehyde addition or model wine without acetaldehyde. Precipitation was observed after the reaction period and increased in response to both higher tannin molecular mass and acetaldehyde concentration. To confirm whether acetaldehyde-mediated condensation of tannin and anthocyanin modified the solubility of the nonbleachable pigments formed, HPLC-MS was used for the semiquantitative identification of vinyl derivatives and ethyl-linked adducts in soluble and precipitated materials. It was found that the proportion of vinyl derivatives and ethyl-linked anthocyanin was elevated in tannin precipitates relative to soluble pigmented material. Despite substantial losses of tannin due to precipitation, the resulting nonbleachable pigment concentration and color intensity were higher in wine media containing elevated acetaldehyde, when each tannin size category was considered independently. The results of this study indicated that the development of nonbleachable pigments from larger tannins may be limited when acetaldehyde-mediated condensation with anthocyanin predominates in wine, concomitant with precipitation.

Focus on the role of seed tannins and pectolytic enzymes in the color development of Pinot noir wine

Maceration techniques which promote the extraction of color pigments and tannin from grapes are often sought in Pinot noir winemaking to optimise color stability; alternatively, exogenous grape tannins may be included during fermentation. To examine the effect of seed-derived tannins and the use of pectolytic enzymes on color development in wines, conventional must preparations of Vitis vinifera L. cv Pinot noir grapes were compared with wines made using a supplementary addition of either a commercial seed tannin product or previously fermented seeds, while in a complementary experiment, seeds were sequentially removed during fermentation. After 6 months bottle aging, wines supplemented with either a commercial seed tannin solution (0.4 ​g/L), or fermented seeds (20% w/w seeds) had from 60% to 95% higher tannin concentration than the untreated wine, and up to 60% more monomeric anthocyanins. Conversely, when a third of the seeds were removed from the fermenting wine, the concentration of both tannin and non-bleachable pigments was 20–30% lower than in untreated wines and the wine hue had more red-purple tones. Exploration of the use of pectolytic enzymes in conjunction with seed removal was also found to have a significant impact on wine color parameters. Further insights on the timing of egress of tannin precursors from seeds was obtained from histochemical examination of the seeds that had been removed during alcoholic fermentation.

Preliminary Study of Australian Pinot Noir Wines by Colour and Volatile Analyses, and the Pivot© Profile Method Using Wine Professionals.

The aim of this preliminary study was to identify potential colour components, volatile and sensory attributes that could discriminate Pinot noir wines from five Australian winegrowing regions (Adelaide Hills, Yarra Valley, Mornington Peninsula, Northern and Southern Tasmania). The sensory analysis consisted of the Pivot© Profile method that was performed by wine professionals. A headspace solid-phase microextraction-gas chromatography-mass spectrometry method was used to quantify multiple volatile compounds, while the Modified Somers method was used for colour characterisation. Analysis of data suggested ethyl decanoate, ethyl 2-methylpropanoate, ethyl 2-methylbutanoate, in addition to decanoic acid as important contributors to the discrimination between regions. Similarly, wine hue, chemical age indices, total anthocyanin, and (%) non-bleachable pigment also discriminated wines between regions. The sensory analysis showed that wines from Mornington Peninsula were associated with the ‘red fruits’ aroma, ‘acidic’, and ‘astringency’ palate descriptors, while those from Adelaide Hills were associated with the ‘brown’ colour attribute. This study indicates regionality is a strong driver of aroma typicity of wine.

Grape skins as supplements for color development in Pinot noir wine

A particular challenge to making wine from Pinot noir grapes is the delicate flavor, light color and poor ageing potential of the wine. Conventional Pinot noir must preparations were compared with those made using a skin-based supplement to assess the impact on non-bleachable (sulfur resistant) pigments in the wine. When supplemented with either fresh grape pomace of Pinot noir, Pinot gris or Chardonnay grapes; Pinot noir grape marc or a commercial liquid grape skin extract, the additional seeds and pulp from the supplements were shown to compromise the development of stable pigments in the wine. To compare the relative merits of tannin derived from grape skins and seeds, the supplements used in a parallel experiment were the skins alone of the same three grape varieties and at six months bottle age, the stable pigment concentration was found to exceed the amount attributable to the supplement. A third experiment used fermented grape skins as the supplement, with 85% of the supplementary anthocyanin recovered as stable pigment complexes in the wine. Notably, this series of experiments showed that supplements containing grape seeds appeared to compromise non-bleachable pigment formation in the wine while skin only supplements stimulated their development.

Impact of Increasing Levels of Oxygen Consumption on the Evolution of Color, Phenolic, and Volatile Compounds of Nebbiolo Wines.

Since the end of the last century, many works have been carried out to verify the effect of controlled oxygen intake on the chemical and organoleptic characteristics of red wines. In spite of the large number of studies on this subject, oxygen remains a cutting-edge research topic in oenology. Oxygen consumption leads to complex and not univocal changes in wine composition, sometimes positive such as color stabilization, softening of mouthfeel, increase of aroma complexity. However, the variability of these effects, which depend both on the oxygenation conditions and the composition of the wine, require more efforts in this research field to effectively manage wine oxygen exposure. The present study is focused on the evolution of the chemical composition of four different Nebbiolo wines, each of them added with 4 different doses of oxygen (7, 14, 21, and 28 mg/L total intake) during the first month of storage. In this perspective, the evolution over time of wine color and polyphenols was studied. Acetaldehyde, glyceraldehyde and glyoxylic acid were quantified by HPLC. These compounds can play a role in wine aging creating condensed colored and stable products involving anthocyanins with or without tannins. Moreover, some volatile aldehydes correlated with oxidized olfactory notes, including methional and (E)-2-alkenals, have been quantified by GC-MS. Overall, during storage a decrease of color intensity, total and free anthocyanins and an increase in polymeric pigments (in particular the contribution to the red color of pigments not-bleachable by SO2 or dTAT%) and some minor aldehydes was observed. Nevertheless, the differences in color parameters between the samples with different doses of oxygen were modest. These evidences were in contrast with an evident and detectable increase of free acetaldehyde content at increasing doses of oxygen measured after 60 days of storage. The effect of oxygen on color and production of SO2 non-bleachable pigments during aging varies with wine composition, with Nebbiolo wines appearing not very reactive in this respect, probably due to their low content in anthocyanins and high content in tannins.

Accentuated Cut Edges (ACE): Effects of Skin Fragmentation on the Composition and Sensory Attributes of Pinot noir Wines

The phenolic composition, aroma, and sensory profiles were evaluated for Pinot noir wines made using four different maceration techniques that modified the floating pomace cap during fermentation: 1) daily plunging; 2) reduced skin particle size (accentuated cut edges, ACE); 3) submerged cap; and 4) ACE plus submerged cap. Throughout vinification, wines were analyzed using rapid analytical techniques to assess the following phenolic attributes: anthocyanin, tannin, nonbleachable pigments, color density, and hue. At six months bottle age (230 days postinoculation), sensory and aroma analyses were conducted on the finished wines. ACE macerated wines were found to have the highest proportion of red color, tannin, nonbleachable pigments, fruit and floral aromas, bitterness, and astringency. Submerging the pomace cap resulted in a lower concentration of phenolic components when compared to ACE wines, but resulted in a significantly higher phenolic content and dark cherry aromas and flavor when compared to the control wine. These findings suggest that the employment of either maceration technique has the potential to make a considerable difference to the wine style produced from a given parcel of fruit and may provide an opportunity to press wines earlier in the fermentation. Linear regression analyses were conducted to compare descriptive wine parameters with instrumental phenolic measurements and demonstrated several strong correlations: red color appearance was correlated with both color density (r2 = 0.95) and nonbleachable pigment (r2 = 0.95); dark fruit flavor was correlated with both color density (r2 = 0.85) and nonbleachable pigment (r2 = 0.85); and astringency was correlated with both tannin (r2 = 0.97) and nonbleachable pigment (r2 = 0.87), demonstrating that techniques of rapid chemical analysis were able to provide valuable insights into the sensory properties of the wine and may become useful tools for monitoring the development of the wine during vinification. While submerged cap vinification also increased the tannin and nonbleachable pigment profiles of the wine, ACE maceration was found to be significantly more effective and is likely to be more readily adapted for application in commercial wineries.

Interactions of Grape Skin, Seed, and Pulp on Tannin and Anthocyanin Extraction in Pinot noir Wines

Berry tissue components (skin, seed, and pulp) were isolated from Pinot noir grapes to determine the tannin content of each component. In addition, the role of each berry component in determining the phenolic profile of the wine was investigated by omitting or doubling each berry tissue in the must and fermenting these using submerged cap micro-vinification. Monomeric anthocyanin, total tannin and non-bleachable pigment concentrations in juice and wine were examined at seven time intervals from yeast inoculation (day 0) to 12 months bottle age (day 400). When included in the must, berry pulp was associated with reduced wine tannin concentration, an effect that was more selective for seed tannin. Consequently the tannin composition of wines made with whole-berry ferments most closely reflected that of skin tannin. The formation of non-bleachable pigments was greater in treatments where seeds were present during fermentation. However, when the contribution of seed tannin in wine was increased by doubling the complement of seeds in the must, anthocyanin concentration remained constant and non-bleachable pigments did not increase during ageing. Treatments with a double complement of grape skins were found to enhance both non-bleachable pigment and tannin in aged wines.

Yeast strain affects phenolic concentration in Pinot noir wines made by microwave maceration with early pressing.

This study examined the effects of yeast strains in a novel winemaking process that had been designed to optimize phenolic extraction and improve production efficiency for Pinot noir winemaking. Microwave maceration with early pressing and co-inoculation of yeast and malolactic bacteria for simultaneous alcoholic and malolactic fermentation was investigated. Yeast treatments (Saccharomyces cerevisiae RC212 and EC1118, and Saccharomyces bayanus AWRI1176) were co-inoculated with Oenococcus oeni PN4 immediately after must microwave maceration. Alcoholic and malolactic fermentation were complete 17 days postinoculation for all three yeast treatments. At 16-month bottle age, the AWRI1176-treated wines had approximately twice the nonbleachable pigment and colour density of wines fermented by EC1118 and RC212. The novel winemaking process produced Pinot noir wine that was stable 37 days after fruit had been harvested and yeast strain choice significantly impacted the stability and phenolic character of wine. Successful simultaneous alcoholic and malolactic fermentation in 17 days, and a demonstrated lack of inhibition between the yeast strains and malolactic strain applied in this study, provide proof of concept for very rapid red winemaking using the novel winemaking approach described herein. Further investigation would be required to assess strain effects on wine aroma, mouth feel and taste, however, this novel winemaking approach may offer significant industry efficiencies.

Properties of wine polymeric pigments formed from anthocyanin and tannins differing in size distribution and subunit composition.

To explore the effect of tannin composition on pigment formation, model ferments of purified 3-O-monoglucoside anthocyanins (ACN) were conducted either alone or in the presence of two different tannins. Tannins were isolated from grape seeds (Sd) or skins (Sk) following exhaustive extraction in 70% v/v acetone. The Sd and Sk tannin fractions had a mean degree of polymerization of 5.2 and 25.6, respectively. The Sd fraction was highly galloylated, at 22%, but galloylation was <2% in the Sk fraction. The Sk fraction was distinguished by a high proportion of prodelphinidin, at 58%. After a 6 month aging period, polymeric pigments were quantified and their color properties determined following isolation by solid-phase extraction. Wine color and polymeric pigment were highest in the treatment containing ACN+Sd and similar in the ACN+Sk and ACN treatments. The same trend between treatments was observed for total and polymeric nonbleachable pigments. Only minor changes in tannin subunit composition were found following ACN incorporation, but the size distribution of polymeric pigments determined by gel permeation chromatography decreased, in particular for the ACN+Sk treatment. Color incorporation in the higher molecular mass range was lower for ACN+Sk wines than for ACN+Sd wines. Compositional differences between the two tannin fractions may therefore limit the incorporation of ACNs in the colored form. The results suggest that in the ACN+Sk and ACN treatments, the formation of lower molecular mass oligomeric pigments was favored. In polymeric pigments derived from ACNs, the presence of ethyl- and vinyl-linked ACNs to the level of trimers was identified using mass spectrometry.

Development of wine colour and non-bleachable pigments during the fermentation and ageing of (Vitis vinifera L. cv.) Cabernet Sauvignon wines differing in anthocyanin and tannin concentration

Non-bleachable (SO2-resistant) pigment formation in wine is related to the concentration of grape-derived anthocyanin and tannin, but their relative impact on the formation and composition of SO2-resistant pigments is poorly understood. Colour development during fermentation and ageing was analysed in two wines produced from grape batches with different anthocyanin and tannin concentrations. Higher anthocyanin and tannin concentrations extracted during fermentation were associated with increased wine colour density, total SO2-resistant pigments and SO2-resistant polymeric pigment. During ageing, wine colour density decreased, but differences between the respective wine treatments were retained. Wine tannin concentration was unchanged with age, anthocyanin decreased, and SO2-resistant pigments increased. Fractionation of wines indicated that SO2-resistant pigments in both polymeric and non-polymeric forms increased with ageing. Increases in tannin pigmentation over time were associated with decreased tannin molecular mass, reflecting the conversion of tannin to lower molecular mass pigmented polymers. The formation of polymeric pigment during ageing was related to the initial wine tannin concentration. However, non-polymeric forms of SO2-resistant pigments increased to a greater extent in wines with higher initial anthocyanin and tannin concentrations. The data suggests that both polymeric and low molecular mass anthocyanin derivatives are important for stable wine colour development.

Microwave Maceration with Early Pressing Improves Phenolics and Fermentation Kinetics in Pinot noir

Microwave maceration with early pressing was applied to Pinot noir must and produced wine with faster fermentation kinetics and significantly higher yeast assimilable nitrogen than control must and 10% greater juice yield than heat macerated must. UV-visible spectrophotometry showed microwave maceration wine as equivalent to or greater than control wine for total pigment, anthocyanin, tannin, and nonbleachable pigment concentration. Microwave maceration was compared with heat maceration, and phenolic differences apparent in juice were not sustained in bottle-aged wines. Histological examination of grape skins showed more substantial intracellular damage in microwave macerated skins than heat macerated and control skins. The results suggest microwave maceration with early pressing warrants serious consideration as an efficient production process for phenolic-rich Pinot noir wine.

Yeast Effects on Pinot noir Wine Phenolics, Color, and Tannin Composition

Extraction and stabilization of wine phenolics can be challenging for wine makers. This study examined how yeast choice affected phenolic outcomes in Pinot noir wine. Five yeast treatments were applied in replicated microvinification, and wines were analyzed by UV-visible spectrophotometry. At bottling, yeast treatment Saccharomyces cerevisiae RC212 wine had significantly higher concentrations of total pigment, free anthocyanin, nonbleachable pigment, and total tannin and showed high color density. Some phenolic effects were retained at 6 months' bottle age, and RC212 and S. cerevisae EC1118 wines showed increased mean nonbleachable pigment concentrations. Wine tannin composition analysis showed three treatments were associated with a higher percentage of trihydroxylated subunits (skin tannin indicator). A high degree of tannin polymerization was observed in wines made with RC212 and Torulaspora delbruekii , whereas tannin size by gel permeation chromatography was higher only in the RC212 wines. The results emphasize the importance of yeast strain choice for optimizing Pinot noir wine phenolics.

Phenolic Compositions of 50 and 30 Year Sequences of Australian Red Wines: The Impact of Wine Age

The phenolic composition of red wine impacts upon the color and mouthfeel and thus quality of the wine. Both of these characteristics differ depending on the age of a wine, with the purple of young wines changing to brick red and the puckering or aggressive astringency softening in older wines. This study investigated the color parameters, tannin concentrations and tannin composition of a 50 year series of Cabernet Sauvignon wines from a commercial label as well as 30 year series of Cabernet Sauvignon and Shiraz wines from a separate commercial label to assess the impact of wine age on phenolic composition and concentration. The wine color density in wines of 40 to 50 years old was around 5 AU compared with 16 AU of wine less than 12 months old, which correlated well with the concentration of non-bleachable pigments and pigmented polymers. Conversely, the anthocyanin concentrations in 10 year old wines were substantially lower than that of recently bottled wines (around 100 mg/L compared with 627 mg/L, respectively), adding further evidence that non-bleachable pigments including pigmented polymers play a much larger role in long-term wine color than anthocyanins. No age-related trend was observed for tannin concentration, indicating that the widely noted softer astringency of older red wines cannot necessarily be directly related to lower concentrations of soluble wine tannin and is potentially a consequence of changes in tannin structure. Wine tannins from older wines were generally larger than tannins from younger wines and showed structural changes consistent with oxidation.

Relationship between Red Wine Grade and Phenolics. 2. Tannin Composition and Size

Commercial red wines ( Vitis vinifera L. cv. Shiraz) produced during the 2009 vintage underwent winemaker assessment for allocation grade soon after production. The wines were then subjected to phenolic analysis to measure wine color (total anthocyanin, SO(2) nonbleachable pigment, and wine color density) and tannins (concentration, composition, and average degree of polymerization). A positive relationship was found between wine phenolic concentration and projected bottle price. Tannin compositional analysis suggested that there was specifically a relationship between wine grade and skin-derived tannins. These results suggest that maximization of skin tannin concentration and/or proportion is related to an increase in projected wine bottle price.

The effect of Saccharomyces cerevisiae and Saccharomyces bayanus yeast on colour properties and pigment profiles of a Cabernet Sauvignon red wine

Red wine was made from the same batch of Cabernet Sauvignon grapes but with two different yeasts, Saccharomyces cerevisiae (SC) or Saccharomyces bayanus (SB). The colour properties and pigment profiles of SC and SB wines were compared at 8 days and 387 days after yeast inoculation. Grape anthocyanin concentration was found to be lower in SB wines than in SC wines at day 8 and 387, but SB wine exhibited greater wine colour density, a* and b*. The anthocyanin concentration did not correlate with wine colour density. The levels of pigmented polymers and SO2 non-bleachable pigments were found to be higher in SB wine at day 387, demonstrating that the formation of stable pyranoanthocyanins and pigmented polymers was enhanced by SB yeast. Electrospray mass spectrometry was used for the compositional analysis of pyranoanthocyanins, and direct and ethyl-bridged linked condensation dimers of malvidin 3-O-glucoside (MvG) with flavanols (catechin or epicatechin). The data demonstrated that the formation of acetaldehyde-mediated pigments was enhanced by the use of the SB yeast. The compositional analysis suggested that the differences in colour properties and pigment profiles of SC and SB wines were largely due to the greater production of acetaldehyde-mediated pigments by the use of SB yeast.

Effect of SO2Concentration on Polyphenol Development during Red Wine Micro-oxygenation

A Merlot wine in 15 L research tanks was subjected to micro-oxygenation at 10 mL O2 per liter of wine per month over a 16 week period with additions of 0, 50, 100, and 200 mg/L SO2. A large decrease in monomeric anthocyanins and flavan-3-ols was seen in wines with a lower concentration of SO2, coupled with an increase in nonbleachable pigments; an increase in tannin, measured using precipitation with methyl cellulose; and a greater size and red coloration of a proanthocyanidin extract obtained using Sephadex LH-20. These changes were largely suppressed in wines initially treated with 200 mg/L SO2 and occurred more slowly in wines stored in bottles in the absence of O2. The concentration of SO2 is shown to regulate the polyphenol chemistry involved in the formation of polymeric pigments and changes in tannin structure affecting wine astringency.

A Nonbleachable Rhodopsin Analogue with a Slow Photocycle

Archaeal rhodopsins, e.g. bacteriorhodopsin, all have cyclic photoreactions. Such cycles are achieved by a light-induced isomerization step of their retinal chromophores, which thermally re-isomerize in the dark. Visual pigment rhodopsins, which contain in the dark state an 11-cis retinal Schiff base, do not share such a mechanism, and following light absorption, they experience a bleaching process and a subsequent release of the photo-isomerized all-trans chromophore from the binding pocket. The pigment is eventually regenerated by the rebinding of a new 11-cis retinal. In the artificial visual pigment, Rh(6.10), in which the retinal chromophore is locked in an 11-cis geometry by the introduction of a six-member ring structure, an activated receptor may be formed by light-induced isomerization around other double bonds. We have examined this activation of Rh(6.10) by UV-visible and FTIR spectroscopy and have revealed that Rh(6.10) is a nonbleachable pigment. We could further show that the activated receptor consists of two different subspecies corresponding to 9-trans and 9-cis isomers of the chromophore. Both subspecies relax in the dark via separate pathways back to their respective inactive states by thermal isomerization presumably around the C(13)=C(14) double bond. This nonbleachable pigment can be repeatedly photolyzed to undergo identical activation-relaxation cycles. The rate constants of these photocycles are pH-dependent, and the half-times vary between several hours at acidic pH and about 1.5 min at neutral to alkaline pH, which is several orders of magnitude longer than for bacteriorhodopsin.

Specific reaction of 9-cis-retinoyl fluoride with bovine opsin.

Opsin readily undergoes Schiff base formation between an active site lysine and 9-cis- or 11-cis-retinaldehyde to form the visual pigments isorhodopsin (lambda max = 487 nm) and rhodopsin (lambda max = 500 nm), respectively (Dratz, 1977). It would be predicted that 9-cis-retinoyl fluoride (1), an isostere of 9-cis-retinal, should be an active site directed, mechanism-based labeling agent of opsin, since a stable peptide bond should be formed instead of a Schiff base. It is shown here that 9-cis-retinoyl fluoride (1) reacts with opsin in a time-dependent fashion (t1/2 = 9 min at 25 microM 1) to form a new, nonbleachable pigment with a lambda max of approximately 365 nm. beta-Ionone competitively slows down the rate of the reaction. The absorbance of the new pigment at approximately 365 nm is similar to that of model amide compounds. This result is consistent in a general and qualitative way with the Nakanishi-Honig point-charge model for visual pigments which requires that the chromophore be charged, a situation not possible when the retinoid is linked to opsin via a peptide bond rather than a protonated Schiff base [Honig, B., Dinur, U., Nakanishi, K., Balogh-Nair, V., Gawinowicz, M.A., Arnabaldi, M., & Motto, M.G. (1979) J. Am. Chem. Soc. 101, 7084-7086]. 9-cis-Retinoyl fluoride (1) is approximately 4-fold more potent than all-trans-retinoyl fluoride (2) as an inactivator of bovine opsin. Importantly, 13-cis-retinoyl fluoride (3) is inactive, and no new absorption band at 365 nm is observed.(ABSTRACT TRUNCATED AT 250 WORDS)