Identification of enzyme coupling sites with aromatic diazonium salts-a resonance raman study.

The coupling reaction of diazonium salts of aromatic compounds with the aromatic residues of proteins results in chromophoric covalent derivatives which yield strong resonance enhanced Raman spectra. The protein residues modified by these coupling reactions have been identified using the ν(NN) and ν(N-φ) vibrational bands in the resonance Raman spectra. Previous studies have established that diazoarsanilic acid couples with carboxypeptidase at tyrosine 248. The resonance Raman spectrum of arsanilazocarboxypeptidase was compared with spectra of arsanilazotyrosine and arsanilazohistidine model compounds; the results are consistent only with coupling at a tyrosine residue. This confirmation of the previously established site of modification establishes the utility of resonance Raman spectroscopy as a tool for identification of the site of covalent modification. To further investigate this approach, the diazonium salt of sulfanilamide (a site-specific reagent) was used to prepare a covalent coupling derivative of bovine carbonic anhydrase. The coupling reaction appears to have a stoichiometry of 1:1 and results in nearly complete loss of sulfanilamide binding capability and esterase activity. Comparison of the pH dependence of the resonance Raman spectra of sulfanilazocarbonic anhydrase with the spectra of sulfanilazotyrosine, sulfanilazohistidine, and sulfanilazotryptophan suggests that histidine is the site of modification of this new carbonic anhydrase derivative.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. V. pH-dependent conformational changes in peptides containing two adjacent tryptophyl residues as evidenced by circular dichroism studies.

The influence of pH upon CD spectra of H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, and H-Gly-Trp-Trp-OH is investigated and data are compared with those obtained for peptides containing only one tryptophyl residue. A negative Cotton effect at around 225 nm, which in previous work has been related to an increase of the conformational rigidity in compounds having the sequence -CO-Trp-Trp, is also observed in the case of H-Trp-Trp-OH and H-Trp-Trp-Gly-OH upon deprotonation of the terminal α-amino group. These data, together with observations arising from solvent and temperature effects, give evidence that H-Trp-Trp-OH undergoes a conformational change upon going from acid to alkaline conditions, where the two aromatic side chains become conformationally more rigid relative to each other. This rigidity generates an exciton coupling between the B_b transitions of the two indoles. Hydrophobic forces, including stacking interactions, do not appear important in stabilizing this conformationally rigid structure. Rather, intramolecular electrostatic interactions (e.g., hydrogen bondings or polar interactions between the aromatic side chain and the peptide backbone) as well as interactions with the OH group(s) of the solvent, are suggested to be the salient forces. Possible structures which obey these requisites are discussed.     

Halogenation of aromatic compounds: thermodynamic, mechanistic and ecological aspects

Biological halogenation of aromatic compounds implies the generation of reducing equivalents in the form of e.g. NADH. Thermodynamic calculations show that coupling the halogenation step to a step in which the reducing equivalents are oxidized with a potent oxidant such as O₂ or N₂O makes the halogenation reaction thermodynamically feasible without the input of additional energy in the form of e.g. NADH. In a current model on the halogenation of tryptophan to 7-chloro-l-tryptophan NADH and O₂ are proposed as co-substrates in a reaction in which the aromatic compound is oxidized via an epoxide as intermediate. The thermodynamic calculations thus indicate that such a route hinges on mechanistic insights but has no thermodynamic necessity. Furthermore the calculations suggest that halogenation of tryptophan and other aromatic compounds should be possible with N₂O, and possibly even with nitrate replacing O₂ as the oxidant.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. VI. Circular dichroism studies of Co-oligopeptides of l-phenylalanine, L-tryptophan, and glycine.

The circular dichroic properties of H-Gly-Phe-(Gly)_n-Trp-Gly-OH (II, n = 0,1,2) and of related simpler peptides, such as H-Phe-Gly-OH, H-Gly-Phe-OH, H-Gly-Phe-Gly-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, and H-Gly-Phe-Trp-OH in water and trifluoroethanol solutions are investigated. Peptides containing only one phenylalanyl residue show markedly different near-uv dichroic signals depending on whether this residue is in the N-terminal position or not. The possible origin of this feature is discussed. The study of the oligopeptides II (n = 0,1,2) shows that no strong intramolecular interaction between the two aromatic rings is present. However, the dichroic properties of II (n = 0) are clearly anomalous, and the analysis of H-Gly-Phe-Trp-OH, H-Phe-Trp-Gly-OH, and of H-Phe-Trp-OH at different pH's, confirms that the presence of two adjacent aromatic residues may bring about chiroptical properties which indicate a restriction in the conformational equilibrium of the molecule. The limits, and the possible generalization of this finding, are discussed.     

Synthesis and bioelectrochemical behavior of aromatic amines

Four aromatic amines 1-amino-4-phenoxybenzene (A₁), 4-(4-aminophenyloxy) biphenyl (A₂), 1-(4-aminophenoxy) naphthalene (A₃) and 2-(4-aminophenoxy) naphthalene (A₄) were synthesized and characterized by elemental, spectroscopic (FTIR, NMR), mass spectrometric and single crystal X-ray diffraction methods. The compounds crystallized in monoclinic crystal system with space group P2₁. Intermolecular hydrogen bonds were observed between the amine group and amine/ether acceptors of neighboring molecules. Electrochemical investigations were done using cyclic voltammetry (CV), square wave voltammetry (SWV) and differential pulse voltammetry (DPV). CV studies showed that oxidation of aromatic amines takes place at about 0.9 V (vs. Ag/AgCl) and the electron transfer (ET) process has irreversible nature. After first scan reactive intermediate were generated electrochemically and some other cathodic and anodic peaks also appeared in the succeeding scans. DPV study revealed that ET process is accompanied by one electron. DNA binding study of aromatic amines was performed by CV and UV–visible spectroscopy. These investigations revealed groove binding mode of interaction of aromatic amines with DNA.     

Ortho-metalation, rotational isomerization, and hydride-hydride coupling at rhenium (V) polyhydride complexes stabilized by aromatic amine ligands

An ortho-metalated rhenium (V) polyhydride complex has been prepared through the reaction of ReH₇(PPh₃)₂ with 2-phenylpyridine. Additionally, a small series of neutral rhenium (V) pentahydride complexes, each of which is stabilized by an aromatic amine ligand, has been prepared. E and Z rotational isomers of the ReH₅(PPh₃)₂(aromatic amine) complexes have been observed at low temperatures by NMR spectroscopy. The E and Z rotational isomers arise from a combination of the lack of a mirror plane symmetry element orthogonal to the aromatic ring in the aromatic amine ligands and the restricted rotation about the Re-N bond in such complexes. Restricted rotation about the Re-N bond in the related complex, ReH₅(PPh₃)₂(Py) has previously been observed by Crabtree et al. The restricted rotation about the Re-N bond seems to result from π-donation of the lone electron pair on the rhenium (V) center to the π∗ system of the aromatic amine ligands. Different populations of the E and Z rotational isomers arise from interactions of substituents on the aromatic ring with the other ligands bound to rhenium. The values of ΔG^‡ for the restricted rotation about the Re-N bonds, for the complexes containing 4-phenylpyrimidine, 2-aminopyrimidine, or 2-aminopyridine, range from 9.9 to 11.3 kcal/mole. One of the new compounds reported herein, ReH₅(PPh₃)₂[1-(2-NH₂Pyr)] is the first rhenium (V) polyhydride complex to display hydride-hydride coupling in its ¹H NMR spectrum.     

Cooligopeptides containing aromatic residues spaced by glycyl residues. IX. Fluorescence properties of tryptophan-containing peptides

The fluorescence properties of several cooligopeptides of glycine, phenylalanine, and tryptophan, containing one or two aromatic residues, are investigated. In particular, a detailed analysis is made of the influence of pH upon the quantum yield and the position of the emission maximum (λ_max) in H-Trp-Trp-OH, H-Trp-Gly-OH, H-Gly-Trp-OH, H-Gly-Trp-Gly-OH, H-Trp-Trp-OH, H-Trp-Trp-Gly-OH, H-Gly-Trp-Trp-OH, H-Phe-Trp-OH, H-Phe-Trp-Gly-OH, H-Gly-Phe-Trp-OH, and H-Gly-X-(Gly)_n-Trp-Gly-OH, with X = Phe or Trp, and n = 0,1,2. It is shown that raising the pH from ca. 2 to 11 results in a red shift of λ_max, and an increase in the quantum yield. These changes, mostly structure dependent, are in most cases attributable to electronic perturbations acting directly upon the λ_max of the fluorophore(s) and upon the quenching efficiency of the free amino and carbonyl groups. For the compounds having two adjacent tryptophyl residues, it is shown that the two fluorophores do not appear to have the same emission properties and the quantum yield is lower than expected. The causes of this behavior are discussed in terms of conformational effects, stacking interactions, and radiationless energy transfer. Finally, an attempt is made to correlate fluorescence data with previous circular dichroism data which had indicated the occurrence of a conformationally rigid structure for some of the compounds having two adjacent aromatic residues.     

Prenylation of aromatic compounds,a key diversification of plant secondary metabolites

Prenylation plays a major role in the diversification of aromatic natural products, such as phenylpropanoids, flavonoids, and coumarins. This biosynthetic reaction represents the crucial coupling process of the shikimate or polyketide pathway providing an aromatic moiety and the isoprenoid pathway derived from the mevalonate or methyl erythritol phosphate (MEP) pathway, which provides the prenyl (isoprenoid) chain. In particular, prenylation contributes strongly to the diversification of flavonoids, due to differences in the prenylation position on the aromatic rings, various lengths of prenyl chain, and further modifications of the prenyl moiety, e.g., cyclization and hydroxylation, resulting in the occurrence of ca. 1000 prenylated flavonoids in plants. Many prenylated flavonoids have been identified as active components in medicinal plants with biological activities, such as anti-cancer, anti-androgen, anti-leishmania, and anti-nitric oxide production. Due to their beneficial effects on human health, prenylated flavonoids are of particular interest as lead compounds for producing drugs and functional foods. However, the gene coding for prenyltransferases that catalyze the key step of flavonoid prenylation have remained unidentified for more than three decades, because of the membrane-bound nature of these enzymes. Recently, we have succeeded in identifying the first prenyltransferase gene SfN8DT-1 from Sophora flavescens, which is responsible for the prenylation of the flavonoid naringenin at the 8-position, and is specific for flavanones and dimethylallyl diphosphate (DMAPP) as substrates. Phylogenetic analysis showed that SfN8DT-1 has the same evolutionary origin as prenyltransferases for vitamin E and plastoquinone. A prenyltransferase GmG4DT from soybean, which is involved in the formation of glyceollin, was also identified recently. This enzyme was specific for pterocarpan as its aromatic substrate, and (?)-glycinol was the native substrate yielding the direct precursor of glyceollin I. These enzymes are localized to plastids and the prenyl chain is derived from the MEP pathway. Further relevant genes involved in the prenylation of other types of polyphenol are expected to be cloned by utilizing the sequence information provided by the above studies.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. III. Co-oligopeptides of L-phenylalanine, L-tryptophan, and Glycine: synthesis and ultraviolet absorption properties.

The preparation of the co-oligopeptides of the series H-Gly-Phe-(Gly)_n-Trp-Gly-OH (n = 0, 1, 2) and of other free peptides of glycine, L -tryptophan, and L -phenylalanine is reported. The syntheses have been carried out by conventional methods, using N-hydroxysuccinimide esters for the coupling steps. The ultraviolet absorption properties of the free peptides have been investigated in water. No hypo- or hyperchromicity was found for the aromatic chromophores, with the exception of H-Gly-Phe-Trp-OH, which shows a small but significant hypochromicity. The contribution of the peptide bond to the molar absorptivity in the far ultraviolet has been separated from that of the side chain plus the ? COO^? group by plotting the measured molar absorptivity ? of the farthest accessible uv maximum as a function of the number of peptide bonds (n_A). The peptide bond contribution proved to be independent of n_A in the range n_A = 1–5, thus ruling out the onset of helical conformations in the longer chain peptides.     

Stereoselective oxidation of aromatic sulfides and sulfoxides in the binding domain of bovine serum albumin

The oxidation of aromatic sulfides with achiral oxidizing agents, e.g., sodium metaperiodate (NaIO₄) and hydrogen peroxide (H₂O₂) in the binding domain of bovine serum albumin (BSA), furnished a strong asymmetric bias (max 81%) of the product sulfoxides in fairly high chemical yields. The kinetic resolution of racemic aromatic sulfoxides was also carried out in the chiral binding domain, and the remaining unchanged sulfoxides showed optical purities ranging over 1–33% at ca. 50% completion of oxidation. The combination of the two stereoselective oxidations above mentioned produced several optically active sulfoxides of >90% optical purity in ca. 50% chemical yield. The present method constitutes a successful biomimetic approach to achieving stereoselectivities as high as obtained by sulfur-oxidizing microorganisms.     

Bio-oxidation of aliphatic and aromatic high molecular weight alcohols by Pichia pastoris alcohol oxidase

Summary The alcohol-oxidase-mediated oxidation of hexanol to hexanal was conducted by whole cells of Pichia pastoris in a biphasic reaction medium consisting of 3% water and 97% (v/v) water-saturated hexane. At substrate levels of ca. 10 g/l, hexanal was produced at a rate of 0.2 g/g cell dry wt. per hour with product yields and carbon recoveries of 96% or greater. Although the substrate range of P. pastoris alcohol oxidase has been documented as C₁–C₅ aliphatic alcohols and benzyl alcohol, the use of a biphasic organic reaction medium showed that this enzyme can also oxidize higher molecular weight aliphatic alcohols of C₆–C₁₁, as well as the aromatic alcohols phenethyl alcohol and 3-phenyl-1-propanol. The ability of alcohol oxidase to oxidize low-water-soluble alcohols greatly extends the utility of this enzyme.Issued as NRCC no. 30955 Offprint requests to: W. D. Murray     

Co-oligopeptides containing two aromatic residues spaced by glycyl residues. X. Proton magnetic resonance study of co-oligopeptides of tryptophan and glycine

The ¹H-nmr spectra of co-oligopeptides of tryptophan and glycine with structure H-Gly-Trp-(Gly)_n-Trp-Gly-OH (n = 0–2) and those of several di- and tripeptides have been recorded at 360 MHz with CD₃OD solutions containing 0.1N NaOD. The assignment of resonance signals was generally possible by comparing the spectra of structurally related peptides with each other. In order to solve the remaining ambiguities in the assignment, H-(αL,βS)(α,β-d₂)Trp-OH, H-Trp-(αL,βS)(α,β-d₂)Trp-OH, and H-Trp-(δ¹,ε²,ζ²,ζ³,η²-d₅)Trp-OH have been prepared and their spectra compared with those of the undeuterated compounds. The distribution of rotamers around the χ₁ and (in two cases) χ₂ torsion angles of the side chains has been obtained from the vicinal coupling constants ³J and from the long-range coupling constants ⁴J. These data and an analysis of the chemical shifts of the Gly-C^α protons suggest that the orientation of the aromatic side chain is influenced by the following order of decreasing interaction with the functional groups at N- and C-side: -NH₂ > –NHCO– > –CONH–> –COO^?. This rule does not hold for the second Trp residue of di- and tripeptides containing the -Trp-Trp- sequence, which has tentatively been attributed to steric effects.     

The aromatic residue content of the enzyme rhodanese.

The aromatic amino acid composition of the enzyme rhodanese has been redetermined. Previous reports have varied from 5 to 11 tryptophans per 26 alanine residues. The present work has quantitated the aromatic residues by a combination of amino acid analysis, solvent perturbation difference spectroscopy, specific residue modification and direct ultraviolet spectral analysis. These methods indicate that rhodanese contains 10 tyrosines, eight tryptophans and 16 phenylalanines per 26 alanine residues. The results for tyrosine and phenylalanine are in reasonable agreement with previous results.     

Theoretical studies on protein-nucleic acid interactions. III. Stacking of aromatic amino acids with bases and base pairs of nucleic acids

Stacking of aromatic amino acids tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe), and histidine (His) with bases and base pairs of nucleic acids has been studied. Stacking energies of the amino acid–base (or base pair) complexes have been calculated by second-order perturbation theory. Our results show that, in general, the predominant contribution to the total stacking energy comes from the dispersion terms. In these cases, repulsion energy is greater than the sum of electrostatic and polarization energies. In contrast to this, interaction of histidine with the bases and base pairs is largely Coulombic in nature. The complexes of guanine with aromatic amino acids are more stable than the corresponding complexes of adenine. Among pyrimidines, cytosine forms the most stable complexes with the aromatic amino acids. The G · C base pair has the highest affinity with aromatic amino acids among various sets of base pairs. Optimized geometries of the stacked complexes show that the aromatic moieties overlap only partially. The heteroatom of one residue generally overlaps with the other aromatic moiety. There is a considerable degree of configurational freedom in the stacked geometries. The role of stacking in specific recognition of base sequences by proteins is discussed.     

On the stereoselective synthesis of (+)-pinoresinol in Forsythia suspensa from its achiral precursor, coniferyl alcohol

The residue from Forsythia suspensa stems, upon removal of soluble enzymes, has provided the first evidence for a stereoselective coupling enzyme in lignan biosynthesis. This preparation catalyses the preferred formation (ca 65%) of (+)-[8,8'-14C]pinoresinol from [8-14C]coniferyl alcohol in the absence of exogenously provided cofactors; addition of H2O2 had little effect on enantiomeric composition. However, when NAD and malate were supplied, the stereoselectivity of the coupling reaction was significantly enhanced and pinoresinol consisting of ca 80% of the (+)-antipode was obtained. Clearly, the insoluble residue contains a specific coupling enzyme which catalyses (+)-pinoresinol formation from coniferyl alcohol. By contrast, when [8-14C]sinapyl alcohol was employed as substrate, only racemic syringaresinols were formed: this non-stereoselective peroxidase-catalysed coupling reaction presumably accounts for the low levels of (-)-pinoresinol encountered in this system when coniferyl alcohol is used as a substrate.     

Two-dimensional NMR studies of the zinc finger motif: solution structures and dynamics of mutant ZFY domains containing aromatic substitutions in the hydrophobic core.

Solution structures of mutant Zn fingers containing aromatic substitutions in the hydrophobic core are determined by 2D-NMR spectroscopy and distance-geometry/simulated annealing (DG/SA). The wild-type domain (designated ZFY-6) is derived from the human male-associated protein ZFY and represents a sequence motif (Cys-X2-Cys-X-Ar-X7-Leu-X2-His-X4-His) that differs from the consensus (Cys-X2,4-Cys-X3-Phe-X5-Leu-X2-His-X3-His) in the location ("aromatic swap") and diversity (Ar = tyrosine, phenylalanine, or histidine) of the central aromatic residue (underlined). In a given ZFY domain the choice of a particular aromatic residue is invariant among vertebrates, suggesting that alternative "swapped" aromatic residues are functionally inequivalent. 2D-NMR studies of analogues containing tyrosine, phenylalanine, or histidine at the swapped site yield the following results. (i) The three DG/SA structures each retain the beta beta alpha motif and exhibit similar staggered-horizontal packing between the variant aromatic residue and the proximal histidine in the hydrophobic core. (ii) The structures and stabilities of the tyrosine and phenylalanine analogues are essentially identical, differing only by local exposure of polar (Tyr p-OH) or nonpolar (Phe p-H) surfaces. (iii) The dynamic stability of the histidine analogue is reduced as indicated by more rapid protein-deuterium exchange of hydrogen bonds related to secondary structure and amide-sulfur coordination (slowly exchanging amide resonances in D2O) and by more extensive averaging of main-chain dihedral angles (3J alpha NH coupling constants). An aspartic acid in the putative DNA recognition surface, whose configuration is well-defined as a possible helix N-cap in the tyrosine and phenylalanine analogues, exhibits multiple weak main-chain contacts in the NOESY spectrum of the histidine analogue; such NOEs are geometrically inconsistent and so provide complementary evidence for structural fluctuations. (iv) Because the three DG ensembles have similar apparent precision, the finding of reduced dynamic stability in the histidine analogue emphasizes the importance of experiments that directly probe fluctuations at several time scales. Our results provide insight into the design of biological metal-binding sites and the relationship of protein sequence to structure and dynamics.     

Shielding effects of the D-Phe aromatic ring in the 1H NMR spectrum of gramicidine S.

Analysis of the side-chain coupling constants for the D-Phe residue of gramicidin S shows that the rotamer predicted for the minimum-energy structure is the predominant one. The aromatic ring of D-Phe shields the Pro C^δ hydrogens, and the calculated shielding closely matches that found in the ¹H NMR spectrum. As the temperature is raised, the rotamer population changes and the shielding is reduced accordingly. The shielding is absent in the saturated analogue of gramicidin S in which the aromatic rings were hydrogenated without changing the conformation of the rest of the molecule.     

The influences of protonation state of histidine on aromatic/arginine region of aquaporin-1 protein

Aquaporin-1 (AQP1) is widely distributed in the epithelial tissue for water absorption and secretion. The histidine (His182) residue in the aromatic/arginine (ar/R) constriction region plays an important role in transporting water through the membrane. In this study, we have performed a total of 46 ns equilibrium molecular dynamics (MD) simulations, and obtained the influence of His182 in two protonation states (Hsd is the proton at N_δ and Hse is the proton at N_?) on the ar/R region. Water permeation rate shows that it is easier for water molecules to permeate the ar/R region of the AQP1 with residue in the Hsd state than in the Hse state. The minimum radii of the pore in the ar/R region were calculated during the last 10 ns MD simulation. We have analysed the correlation among the state of the pore (open or close), the minimum radius of the ar/R region and the dihedral angles < C_β-C_γ-C_δ-N_? of Arg197. The results show that the minimum radius can be used to mark the state of the pore.     

Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B.

The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from Pseudomonas putida biotype B have been characterized in their roles in steroid binding and catalysis. Kinetic and equilibrium binding analyses were carried out for the mutant enzymes with the substitutions Phe-54 --> Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The removal of their bulky, aromatic side chains at any of these three positions results in reduced k(cat), particularly when Phe-82 or Trp-116 is replaced by Ala. The results are consistent with the binding interactions of the aromatic residues with the bound steroid contributing to catalysis. All the mutations except the F82A mutation increase K(m); the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility that the phenyl ring at position 82 might be unfavorable for substrate binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a phenyl ring at position 82, and a nonpolar aromatic or small side chain at position 116 might be favorable for binding the reaction intermediate. In contrast to the increased K(D) for equilenin, the enzymes with any substitutions at positions 54 and 116 display a decreased K(D) for 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116 might be unfavorable for product binding. The crystal structure of F82A determined to 2.1-A resolution reveals that Phe-82 is important for maintaining the active site geometry. Taken together, our results demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to the stabilization of steroid species including substrate, intermediate, and product.     

Co-oligopeptides of aromatic amino acids and glycine with a variable distance between the aromatic residues. II. Ultraviolet absorption and circular dichroism properties of co-oligopeptides of tryptophan and glycine

The uv absorption and circular dichroism (CD) properties in water (pH 5.9) and trifluoroethanol of several co-oligopeptides of glycine and tryptophan have been investigated. These compounds contain one tryptophyl residue, such as H-Gly-Trp-OH, H-Trp-Gly-OH, and H-Gly-Trp-Gly-OH; or two, such as H-Trp-Trp-OH, H-Gly-Trp-Trp-OH, H-Trp-Trp-Gly-OH, and H-Gly-Trp-(Gly)_n-Trp-Gly-OH (I, n = 0,1,2). Furthermore, the case of some protected derivatives, such as Z-Trp-Gly-OMe, Z-Gly-Trp-Gly-OMe, and Z-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, Z-Gly-Trp-Trp-Ome were investigated. The extinction coefficients in H₂O in the region of the L bands, referred to one mole of tryptophyl residue, are essentially the same for all compounds within the limit of experimental error, thus indicating that no strong chromophoric interaction takes place in the oligopeptides containing two aromatic residues. However, in the far uv region, anomalies in the absorption properties cannot be excluded. The investigated compounds show marked differences in their CD properties. Whereas in the case of lower molecular-weight peptides (e.g., H-Trp-Gly-OH and H-Gly-Trp-OH), these differences can be ascribed, at least in part, to the influence of the charge end groups on the indole chromophore, in the case of the compounds containing two tryptophyl residues, the differences in CD properties are assumed to reflect the different structures and conformations of the oligopeptides. In particular, we observed a negative dichroic band at ～225 nm in H-Gly-Trp-Trp-OH and I(n = 0) in water. In trifluoroethanol, this band has a much larger intensity, and it is present also in Z-Gly-Trp-Trp-OMe, Z-Trp-Trp-Gly-OMe, and Z-Trp-Trp-OMe. It is argued that such a negative band is characteristic of the sequence -CO-Trp-Trp-, and the possibility that it derives from intramolecular chromophoric interaction is discussed. It is also pointed out, particularly in view of the high ellipticity values found in some cases (up to 100,000 deg cm² dmol^?1), that this feature may reflect a high degree of conformational rigidity connected with the sequence -CO-Trp-Trp-.