Isolation, purification and physicochemical characterization of water-soluble Bacillus thuringiensis melanin

Melanins are widely used in medicine, pharmacology, cosmetics and other fields. Although several technologies for the purification of water-insoluble dioxyphenylalanine (DOPA) melanins have been described, a source of water-soluble melanin is highly desirable. Here we describe an effective procedure for the isolation and purification of water-soluble melanin using the culture medium of Bacillus thuringiensis subsp. galleriae strain K1. Water-soluble melanin from this organism has an isoelectric point (pI=3.0-3.2) and was purified optimally by adsorbtion using the IA-1r resin and elution as a concentrated solution. The purified melanin obtained exhibited a similar infra-red absorbtion spectrum to synthetic melanin and contained quinolic and phenolic structures and an amino acid content of around 20% after acid hydrolysis. The molecular weight of the purified melanin determined by SDS-PAGE was 4 kDa and the electromagnetic spin resonance spectrum of the purified microbial melanin was a slightly asymmetric singlet without hyperfine structure with about 7 Gauss width of the line between points of the maximum incline and g=2.006. The concentration of paramagnetic centers in melanin is 0.21x10(18) spin/g. The results obtained provide a rapid, simple and inexpensive method for the large scale purification of water soluble melanin that may have widespread applications.     

Physicochemical characterization and antioxidant activity of melanin from a novel strain of Aspergillus bridgeri ICTF-201

Aims: The aim of the study is to isolate and characterize a melanin pigment from a new strain of Aspergillus bridgeri isolated from rhizosphere soil of Eucalyptus tree and to investigate its antioxidant activity. Methods and Results: The extracellular pigment was alkali soluble, acid‐resistant and insoluble in organic solvents and water. The pigment was precipitated on treatment with FeCl₃, ammoniacal AgNO₃ and potassium ferricyanide and was bleached in the presence of oxidants and reductants. It was confirmed as melanin based on the Fourier transform infrared and electron paramagnetic resonance spectroscopy techniques apart from chemical analysis. Inhibition of melanin production by inhibitors like tricyclazole, 6‐hydroxyflavanone, 4‐hydroxy‐7‐methoxy‐3‐phenyl‐coumarin, 7‐hydroxy‐4‐phenyl‐coumarin and 7‐hydroxy‐3,4,8‐trimethylcoumarin confirmed that melanin produced by A. bridgeri is synthesized by 1,8‐dihydroxynaphthalene (DHN)‐melanin pathway. The melanin showed good free radical scavenging activity by DPPH method with an EC₅₀ of 54·12 μg ml^?1. Conclusions: The results of the study indicate that the melanin produced by the newly isolated A. bridgeri strain is a member of DHN melanin family and exhibited significant free radical scavenging activity. Significance and Impact of the Study: This is the first report on characterization of DHN melanin produced by a novel strain of A. bridgeri and may find potential application as a natural antioxidant in the cosmetic and pharmaceutical industries.     

Melanin Standard Method: Particle Description

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin. There are no standard methods for the isolation, purification, and storage of melanins. Mild methods designed to preserve the native composition and structure of melanin are needed. The specific aim of the present work, using Sepia melanin, was to develop a mild and generally applicable protocol for the isolation and purification of melanins. It is well established that melanin polymers contain a large number of free carboxylic acid residues. These anionic residues are responsible for the cation exchange properties observed for melanins. Heating melanins with hydrochloric acid at reflux has been demonstrated to lead to extensive decarboxylation. Indeed, heat alone has been shown to cause decarboxylation, and care must be exercised to avoid such conditions. By analogy with cation exchange resins, melanins should be isolated and named according to the associated counterion (e.g., Sepia melanin—K⁺ form). The method reported here avoided extremes in pH and temperature, and was designed to yield melanin in the K⁺ form. Physical disaggregation of particulate melanin using a wet milling step was also found to facilitate removal of significant quantities of adsorbed protein. The following physical parameters were used to monitor the purification and to characterize the resultant melanin: pH, conductance, particle size, and diffuse reflectance spectroscopy.     

Aerobic photoreactivity of synthetic eumelanins and pheomelanins: generation of singlet oxygen and superoxide anion

In this work, we examined photoreactivity of synthetic eumelanins, formed by autooxidation of DOPA, or enzymatic oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid and synthetic pheomelanins obtained by enzymatic oxidation of 5‐S‐cysteinyldopa or 1:1 mixture of DOPA and cysteine. Electron paramagnetic resonance oximetry and spin trapping were used to measure oxygen consumption and formation of superoxide anion induced by irradiation of melanin with blue light, and time‐resolved near‐infrared luminescence was employed to determine the photoformation of singlet oxygen between 300 and 600 nm. Both superoxide anion and singlet oxygen were photogenerated by the synthetic melanins albeit with different efficiency. At 450‐nm, quantum yield of singlet oxygen was very low (~10^?4) but it strongly increased in the UV region. The melanins quenched singlet oxygen efficiently, indicating that photogeneration and quenching of singlet oxygen may play an important role in aerobic photochemistry of melanin pigments and could contribute to their photodegradation and photoaging.     

Unexpected impact of esterification on the antioxidant activity and (photo)stability of a eumelanin from 5,6‐dihydroxyindole‐2‐carboxylic acid

To inquire into the role of the carboxyl group as determinant of the properties of 5,6‐dihydroxyindole melanins, melanins from aerial oxidation of 5,6‐dihydroxyindole‐2‐carboxylic acid (DHICA) and its DHICA methyl ester (MeDHICA) were comparatively tested for their antioxidant activity. MALDI MS spectrometry analysis of MeDHICA melanin provided evidence for a collection of intact oligomers. EPR analysis showed g‐values almost identical and signal amplitudes (ΔB) comparable to those of DHICA melanin, but spin density was one order of magnitude higher, with a different response to pH changes. Antioxidant assays were performed, and a model of lipid peroxidation was used to compare the protective effects of the melanins. In all cases, MeDHICA melanin performed better than DHICA melanin. This capacity was substantially maintained following exposure to air in aqueous buffer over 1 week or to solar simulator over 3 hr. Different from DHICA melanin, MeDHICA melanin was proved to be fairly soluble in different water‐miscible organic solvents, suggesting its use in dermocosmetic applications.     

Melanin Standard Method: Titrimetric Analysis

Melanin isolated from the ink sac of Sepia officinalis (Sepia melanin) has been proposed as a standard for natural eumelanin, and a standard mild isolation and purification protocol for Sepia melanin has been developed (Zeise, doctoral dissertation, Johns Hopkins University, 1991). The goal of the present work, developed using Sepia melanin, was to quantify the bioavailable carboxylic acid groups present in melanin particles. Bioavailability is governed by the accessibility of carboxy groups to the surrounding biological milieu, and is expressed as microequivalents of carboxy group per gram of melanin. The present work was carried out using an heterogeneous slurry of melanin in a nonaqueous system. A standard acidic titrant, and an automatic titrator operating in an equilibrium titration mode were used to characterize and quantify the carboxy group content of Sepia melanins and several other commonly used melanins purified by a standard method (Zeise et al., Pigment Cell Res. [Suppl] 2:48–53, 1992).     

Interaction of Melanin with Proteins – The Importance of an Acidic Intramelanosomal pH

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein‐bound form. We also present evidence to show that soluble protein‐bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano–protein complex. The interaction between melanin and proteins appears to be largely charge‐dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein‐bound.     

Characterization of melanin isolated from a dark septate endophyte (DSE), Exophiala pisciphila

Melanin produced by a dark septate endophyte, Exophiala pisciphila, was isolated and purified. The solubility properties, Ultraviolet–visible and Fourier transform infrared spectra of the purified E. pisciphila melanin were similar to those of typical melanin. Inhibition of melanin production was observed when colonies exposed to tricyclazole (1,8-dihydroxynaphthalene (DHN) melanin inhibitor), but not to kojic acid (3,4-dihydroxyphenylalanine melanin inhibitor). Thus the E. pisciphila melanin was a member of DHN melanin family. In addition, the antioxidant activities of E. pisciphila melanin were evaluated in vitro by 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay. E. pisciphila melanin exhibited a strong antioxidant activity. Addition of 50–350 mg L⁻¹ Cd(II) to the medium increased the melanin production in E. pisciphila.     

Purification,crystal structure and antimicrobial activity of phenazine‐1‐carboxamide produced by a growth‐promoting biocontrol bacterium,Pseudomonas aeruginosa MML2212

Aim: To purify and characterize an antimicrobial compound produced by a biocontrol bacterium, Pseudomonas aeruginosa MML2212, and evaluate its activity against rice pathogens, Rhizoctonia solani and Xanthomonas oryzae pv. oryzae. Methods and Results: Pseudomonas aeruginosa strain MML2212 isolated from the rice rhizosphere with wide‐spectrum antimicrobial activity was cultured in Kings’B broth using a fermentor for 36 h. The extracellular metabolites were isolated from the fermented broth using ethyl acetate extraction and purified by two‐step silica‐gel column chromatography. Three fractions were separated, of which a major compound was obtained in pure state as yellow needles. It was crystallized after dissolving with chloroform followed by slow evaporation. It is odourless with a melting point of 220–222°C. It was soluble in most of the organic solvents and poorly soluble in water. The molecular mass of purified compound was estimated as 223·3 by mass spectral analysis. Further, it was characterized by IR, ¹H and ¹³C NMR spectral analyses. The crystal structure of the compound was elucidated for the first time by X‐ray diffraction study and deposited in the Cambridge Crystallographic Data Centre ( http://www.ccde.com.ac.uk ) with the accession no. CCDC 617344 . Conclusion: The crystal compound was undoubtedly identified as phenazine‐1‐carboxamide (PCN) with the empirical formula of C₁₃H₉N₃O. Significance and Impact of the Study: As this is the first report on the crystal structure of PCN, it provides additional information to the structural chemistry. Furthermore, the present study reports the antimicrobial activity of purified PCN on major rice pathogens, R. solani and X. oryzae pv. oryzae. Therefore, the PCN can be developed as an ideal agrochemical candidate for the control of both sheath blight and bacterial leaf blight diseases of rice.     

Retention of the cyanobacterial neurotoxin β‐N‐methylamino‐l‐alanine in melanin and neuromelanin‐containing cells – a possible link between Parkinson‐dementia complex and pigmentary retinopathy

β‐N‐methylamino‐l ‐alanine (BMAA), a neurotoxic amino acid produced by cyanobacteria, has been suggested to be involved in the etiology of a neurodegenerative disease complex which includes Parkinson‐dementia complex (PDC). In PDC, neuromelanin‐containing neurons in substantia nigra are degenerated. Many PDC patients also have an uncommon pigmentary retinopathy. The aim of this study was to investigate the distribution of ³H‐BMAA in mice and frogs, with emphasis on pigment‐containing tissues. Using autoradiography, a distinct retention of ³H‐BMAA was observed in melanin‐containing tissues such as the eye and neuromelanin‐containing neurons in frog brain. Analysis of the binding of ³H‐BMAA to Sepia melanin in vitro demonstrated two apparent binding sites. In vitro‐studies with synthetic melanin revealed a stronger interaction of ³H‐BMAA with melanin during synthesis than the binding to preformed melanin. Long‐term exposure to BMAA may lead to bioaccumulation in melanin‐ and neuromelanin‐containing cells causing high intracellular levels, and potentially changed melanin characteristics via incorporation of BMAA into the melanin polymer. Interaction of BMAA with melanin may be a possible link between PDC and pigmentary retinopathy.     

Heterologous expression and purification of active L‐asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

l ‐asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc_ASNaseI and Sc_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc_ASNaseI (His)₆‐tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni²⁺‐charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc_ASNaseI with high specific activity (110.1 ± 0.3 IU mg^?1) were obtained. In addition, the use of FPLC‐IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 ± 0.01%) was achieved with a purification factor of 17‐fold. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416–424, 2017     

Characterization of a Phenol Oxidase from Cryptococcus neoformans var. neoformans

In Cryptococcus neoformans, enzymic oxidation of various catechols leads to melanin, a proposed virulence factor. A phenol oxidase enzyme of Cryptococcus neoformans var. neoformans produced at 25 C has been purified from an ultracentrifugal supernatant of an extract of broken cells. Hydrophobic interaction chromatography followed by anion-exchange column chromatography allowed purification of the phenol oxidase. The molecular weight of the enzyme estimated by gel filtration was about 80,000 and a dimeric species (Mw = 160,000) was suggested. The isoelectric point of the protein was approximately 4.1. An NH₂-terminal 31 amino acid sequence was determined using phenol oxidase electroblotted onto a PVDF membrane after nondenaturing gel electrophoresis. Upon searching the Peptide Institute (Osaka) data base, no proteins with high degrees of homology were found.     

Improved isolation of glucuronan from algae and the production of glucuronic acid oligosaccharides using a glucuronan lyase

A new source for the production of bioactive glucuronic acid oligosaccharides (GlcUAOs) from the depolymerization of green seaweed Ulva lactuca glucuronan (Algal glucuronan) has been investigated. Algal glucuronan purification was optimized by the acidic precipitation method which allowed us to separate the polysaccharide mixture extracted from the cell wall of Ulva lactuca using hot water containing sodium oxalate. A series of the GlcUAOs were obtained by enzyme degradation of algal glucuronan with a glucuronan lyase (GL) isolated from Trichoderma strain. The putative bioactive GlcUAOs generated were then purified by size-exclusion chromatography in gram quantity and characterized by ¹H/¹³C NMR spectroscopy and ESI-Q/TOF-mass spectrometry.     

Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography

Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion‐exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10⁸ L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:864–871, 2014     

Rapid high‐yield N‐acylation of aminothiols: N‐acetylglutathione and N‐acetylhomocysteine and their thiol pKa values

Methodology for the rapid N‐acylation of aminothiols in aqueous solution using procedures commonly employed in biochemical studies is described here. Glutathione disulfide (GSSG) and homocystine were diN‐acetylated in ~100% yield in 0.1 M aqueous NaHCO₃ (pH 8.5) at room temperature by 2.5 equiv of the activated ester, N‐hydroxysulfosuccinimidyl acetate, an efficient water‐soluble acetylating reagent. Following acetone precipitation, diN‐acetylGSSG was further purified and desalted on a strong anion‐exchange (SAX) cartridge. DiN‐acetylhomocystine was simultaneously purified and desalted on a C₁₈ cartridge. The N‐acetylated aminothiols were generated using gel‐immobilized tris(2‐carboxyethyl)phosphine as a reductant, which obviated the need for further purification. Alternatively, disulfide exchange with dissolved dithiothreitol yielded N‐acetylglutathione, which was purified on the SAX cartridge. pH titrations of N‐acetylglutathione (8.99) and N‐acetylhomocysteine (9.66) as well as those of commercially available N‐acetylcysteine (9.53) and N‐acetylpenicillamine (10.21) yielded pK_a(SH) values of importance for biological studies. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Peptide purification by affinity chromatography based on α‐ketoacyl group chemistry

Significant advances have been achieved in the fields of peptide/protein synthesis, permitting the preparation of large, complex molecules. Shortcomings, however, continue to exist in the area of peptide purification. This paper details some studies we undertook to develop a new strategy for peptide purification based on a reactivity of α‐ketoacyl groups in peptides. The α‐ketoacyl peptide was generated from N^ε‐acyl‐lysyl‐peptide in the solid phase via a transamination reaction using glyoxylic acid and nickel(II) ion. Cleavage of the α‐ketoacyl group with o‐phenylenediamine gave the target peptide in an acceptable yield and purity. We first carried out a careful step‐by‐step optimization of the purification conditions using a model peptide. The strategy was then used in the purification of a transmembrane peptide that could not be effectively purified using a conventional RP‐HPLC system due to the strong hydrophobicity of the peptide and its high tendency to aggregate. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

FIRST EVIDENCE OF PALYTOXIN ANALOGUES FROM AN OSTREOPSIS MASCARENENSIS (DINOPHYCEAE) BENTHIC BLOOM IN SOUTHWESTERN INDIAN OCEAN1

Benthic dinoflagellates of the genus Ostreopsis Schmidt are common in tropical and subtropical water, and some species produce toxins potentially involved in human intoxication events. A benthic bloom of Ostreopsis mascarenensis Quod was observed near Rodrigues Island during a survey of benthic dinoflagellates in the southwestern Indian Ocean. The morphology of O. mascarenensis was studied by LM and SEM. Preliminary screening of a crude extract of an O. mascarenensis bloom revealed neurotoxicity in mice similar to that induced by palytoxin. After partition of the crude extract, the highest toxicity was retained in the butanol‐soluble fraction, which retained hemolytic activity suggestive of palytoxin analogues. Two new toxins, mascarenotoxin‐A and ‐B, were resolved from this fraction by HPLC coupled to a diode array detector. The closed mass spectrum profile and fragmentation pattern obtained by advanced nano–electrospray ionization quadrupole time‐of‐flight mass spectrometry between purified toxins and a reference palytoxin confirmed the mascarenotoxins as palytoxin analogues. These results were confirmed by tandem mass spectrometry with the identification of specific fragment ion m/z 327. An on‐line liquid chromatography protocol coupled to tandem mass spectrometry was developed for detection of these palytoxin analogues. The present study describes the first purification, chemical, and toxicological characterization of new palytoxin analogues isolated from a benthic bloom of O. mascarenensis. These results suggest that O. mascarenensis, which is largely distributed in the southwestern Indian Ocean, could be a source of palytoxin poisoning in this tropical area.     

Structural Studies on “Isosclerotan”, a New Glucan Isolated from Sclerotinia Fungus,by Physical,Chemical and Enzymatic Methods

“Isosclerotan”, a polysaccharide constituent extracted with a sodium hydroxide solution from sclerotia of Sclerotinia libertiana, could be purified by the successive precipitation with the followings; a mixture of copper sulfate and sodium hydroxide, ammonium sulfate, and ethyl alcohol. The preparation proved homogeneous by ultracentrifugal analysis. From sedimentation and viscosity measurements, the molecular weight of isosclerotan was calculated as 6.13 × 10⁶, andas 1.60 × 10⁵ after treatment with a dilute oxalic acid solution. Isosclerotan was scarecely soluble in cold water but soluble in hot water, yielding a highly viscous solution. It exhibited a low positive optical rotation, + 23.0° (in water), and infrared spectrum had a sharp absorption at 890~898 cm^?1, which indicated the prevalence of the β-glycosidic linkage in isosclerotan. Through degradation by acids and enzymes of isosclerotan, there were obtained various oligosaccharides containing β-1.3, β-1.4, and β-1.6 linkages. From results obtained by periodate oxidation and methylation, it is assumed that the polysaccharide involves the 1.3, 1.4, and 1.6 linkages in 47.7%, 16.6% and 35.7%, respectively, and a branching structure about 12.5%.     

A C-NMR study of 10,12-tricosadiynoic acid and the corresponding phospholipid and phospholipid polymer

Diacetylene phospholipids are presently being studied because of their potential to polymerise in vesicles, multilayers and natural biomembranes. ¹³C-NMR spectra and spin-lattice relaxation times have now been obtained of a diacetylene phospholipid present in a sonicated dispersion in water. Similar data have been obtained of a monoacetylene phospholipid and a saturated phospholipid. For further comparison the spectrum of a diacetylenic fatty acid in benzene-d₆ was also examined and relaxation data obtained. A comparison of the various relaxation data provides an indication of the restricted motion associated with the two conjugated triple bonds of the diacetylene phospholipid within the lipid bilayer structure. A proximity interaction between diacetylene groups occurs and a conformation for the diacetylene part of the lipid in the bilayer is deduced. The ¹³C-NMR spectrum of a soluble phospholipid polymer in C²HCl₃, obtained by ultraviolet irradiation of the diacetylene phospholipid, shows that the two conjugated triple bonds of the monomer is replaced in the polymer by an alternating double and triple bonded conjugated structure.