The self-organization of adenosine 5'-triphosphate and adenosine 5'-diphosphate in aqueous solution as determined from ultraviolet hypochromic effects

The self-association of adenosine 5'-triphosphate (ATP) and of adenosine 5'-diphosphate (ADP) was studied in aqueous solution at different pH values, over the concentration range from 5 x 10(-6) to 5 x 10(-2) M, by ultraviolet spectroscopy. Measures of the molar absorptivity of the ultraviolet bands of these compounds with increasing concentration have shown two hypochromic effects, at concentrations below 10(-3) and above 10(-3) M, respectively. These results can be interpreted in terms of self-association processes involving the formation of dimers and of polymers. From the fitting of the experimental curves of hypochromic effects, self-association constants for dimerization and polymerization were calculated. The results obtained are discussed in relation to the values reported in the literature and indicate the influence of the concentration range not only on the numerical value but also on the order of magnitude of the association constants. Comparison of ATP with ADP shows that the length of the phosphate chain may be a relevant feature in the nature of the self-organization processes in these adenine nucleotides in aqueous solution.     

Self-association of human apolipoproteins A-I and A-II and interactions of apolipoprotein A-I with bile salts: quasi-elastic light scattering studies

We employed quasi-elastic light scattering (QLS) to systematically study the aqueous self-association of human apolipoproteins A-I and A-II (apo A-I and apo A-II) and the interactions of apo A-I with common taurine-conjugated bile salts. Self-association of apo A-I was promoted by increases in apolipoprotein concentration (0.09-2.2 mg/mL) and ionic strength (0.15-2.0 M NaCl), inhibited by increases in temperature (5-50 degrees C) and guanidine hydrochloride concentration (0-2.0 M), and unaffected by hydrostatic pressures up to 500 atm. The mean hydrodynamic radius (Rh) of apo A-I micelles ranged from 38 A to a maximum asymptotic value of 68 A. We examined several possible models of apo A-I self-association; the model that best fitted the Rh values assumed that apo A-I monomers first interacted at low concentrations to form dimers, which then further associated to form ring-shaped limiting octamers. Comparison of the temperature-dependent and ionic strength dependent free energy changes for the formation of octamers from apo A-I dimers suggested that hydrophobic forces strongly favored self-association and that electrostatic repulsive forces were only weakly counteractive. Apo A-II self-association was also promoted by increases in apolipoprotein concentration (0.2-1.8 mg/mL) and inhibited by increases in guanidine hydrochloride concentration (0-1.0 M) but was unaffected by variations in temperature (10-37 degrees C): the largest Rh values observed were consistent with limiting tetramers. As demonstrated by equilibrium dialysis, bile salts in concentrations below their critical micellar concentrations (cmc) bound to apo A-I micelles but had no effect upon apo A-I self-association, as inferred from constant Rh values. When bile salt concentrations exceeded their aqueous cmc values, a dissociation of apo A-I micelles resulted with the formation of mixed bile salt/apo A-I micelles. These studies support the concepts that apo A-I and apo A-II form small dimeric micelles at low concentrations that grow sharply to reach limiting sizes over a narrow concentration range. The influences of bile salt concentration and species upon these micelles have relevance to the plasma transport of bile salts in high-density lipoproteins and to the physical-chemical state of apo A-I and apo A-II molecules in native biles.     

Aspects of the molecular structure and dynamics of neuropeptide Y.

Human neuropeptide Y (hNPY) and the Q34-->P34 mutant (P34-hNPY) have been characterized by CD spectroscopy. hNPY self-associates in aqueous solution with a dimerization constant in the micromolar range. The self-association correlates with an increase in secondary-structure content which was studied as a function of concentration, temperature and pH. The effects of temperature were measured in water (5-84 degrees C) and in ethanediol/water (2 : 1) (-90 degrees to +90 degrees C). A single-residue mutation, Q34-->P34, affects the pH, thermal and self-association properties of NPY. The CD results are correlated with photochemically induced dynamic nuclear polarization NMR experiments which show that the tyrosines at the interface between two monomer units present limited accessibility to a photoreactive dye. An equilibrium state is described, involving a PP-fold monomer form and a handshake dimer form, that accommodates the physicochemical properties of NPY.     

A thermodynamic model for the self-association of human spectrin

The self-association of human spectrin at 28.8 degrees C in 0.11 M salt (pH 7.5) has been studied by means of sedimentation equilibrium. Coincidence of omega function plots as a function of total spectrin concentration (0-2 g/L) indicated that equilibrium was achieved and that no significant concentration of solute was incapable of participating in the self-association reaction. On the basis of the root-mean-square deviation of the fits and the randomness of the residuals, the behavior can be described equally well, either by a cooperative isodesmic model, in which K12 approximately 2 x 10(6) M-1 and all other K approximately 10(6) M-1, or by an attenuated scheme in which K(i-1)i approximately (3.5 x 10(6)/i M-1. The returned values of the second virial coefficient, B, for both these models fall within the range calculated from the charge and Stokes radius of spectrin. A mechanism for spectrin self-association consistent with both schemes is proposed in which spectrin heterodimers undergo a reversible opening at the self-association interface. These open heterodimers then undergo indefinite self-association to form a series of open-chain oligomers in dynamic equilibrium with closed-loop oligomers.     

Parallel double helical DNA. 1. Evidence for the existence of a spiral A-T-paired base

The dependence of UV and CD spectra of oligonucleotide 3'-d(ApTpApTpApTpApTpApTp)-O(CH2)6O-5'-(pApTpApTpApTpApTp ApT) (eicosamer) in aqueous solution at pH 7 in the presence of 0.5 M NaCl on temperature and concentration was studied. It was shown that the eicosamer in concentrations below 5.10(-4) M forms a parallel stranded hairpin. From the thermal denaturation profile the thermodynamic parameters of parallel hairpin formation were determined. The values of delta H0, delta S0 and Tm were -90 +/- 8 kJ/mol, -300 +/- 20 J.mol-1.K-1 and 40.5 degrees C, respectively. The CD spectra of the parallel helix differ from those of B-form DNA by reduction of extreme magnitude at approximately 265 nm and appearance of a negative effect at approximately 285 nm.     

Self-association processes involving anthracene labeled phosphatidylcholines in model membrane

When studying lipid-lipid or lipid-protein interaction in membranes, the correct interpretation of data obtained when using fluorescent phospholipid probes requires the best possible knowledge of probe behaviour in phospholipid membranes. Analysis of the translational dynamics and photochemical properties of the anthracene-labeled phosphatidylcholine (EAPC) shows that a self-association process occurs with this probe in the membrane at the ground state. This anthracene self-association is characterized and leads to a hypochromic effect which has been studied by means of ultraviolet absorption spectroscopy in unilamellar egg-yolk phosphatidylcholine (EggPC) vesicles. A model with indefinite linear self-association, in which each step has the same equilibrium constant, best describes the data. The equilibrium constant was found to be in the 300-500 M(-1) range and the complex lateral distribution pattern of EAPC in model membranes, which results from this self-association process, is characterized and seems to be mainly controlled by the amount of EAPC incorporated into the lipid bilayer.     

A spectroscopic investigation of the self-association and DNA binding properties of a series of ternary ruthenium(II) complexes

Six ruthenium(II) complexes of the general form cis-alpha-[Ru(N4-tetradentate)(N2-bidentate)]Cl2 have been synthesized from the two related tetradentate ligands 1,6-di(2'-pyridyl)-2,5-dimethyl-2,5-diazahexane (picenMe2) and 1,6-di(2'-pyridyl)-2,5-dibenzyl-2,5-diazahexane (picenBz2) and the bidentate ligands 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) and dipyrido[3,2-f:2'3'-h]quinoxaline (dpq). Synthetic intermediate species of the general form cis-alpha-[Ru(II)(N4-tetradentate)(DMSO)Cl][PF6] were isolated. The N4-tetradentate ligand picenMe2 formed only the cis-alpha stereoisomer, while picenBz2 formed both the cis-alpha and cis-beta stereoisomers. These latter stereoisomers were resolved by fractional crystallisation. Dimer self-association constants, K(D), were estimated from the concentration dependence of the 1H NMR shifts for some of these complexes in aqueous solutions at 25 degrees C. The values of K(D) ranged from 0.6 to 7.9 M(-1) and a relationship was observed between the aromatic surface area of the bidentate component and the degree to which self-association occurred, whereby a greater level of self-association correlates with a larger surface area for the bidentate ligand. Some of these complexes demonstrate an ability to bind to DNA that is consistent with intercalation of the bidentate molecular component between the base pairs of the DNA molecule. Using calf-thymus DNA, the equilibrium binding constants, K(B), were determined for some of the complexes using intrinsic methods and these ranged from 3.32 to 5.11 M(-1), the intercalating abilities of the different bidentate ligands being in the order dp q > phen > bipy. This relationship between aromatic surface area of the bidentate ligand and the degree of DNA binding activity is the same as that observed in the self-association study.     

One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium

The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.     

Behavior of human apolipoprotein E in aqueous solutions and at interfaces

Human plasma apolipoprotein E (apo-E) strongly self-associates to form a stable tetramer in an aqueous solution at pH 7.4 containing 0.15 M NaCl. Tetramerized apo-E is abundant in alpha-helical conformation with an asymmetric molecular shape. Apo-E forms a stable monolayer at the air-water interface with a collapse pressure of 14 dynes/cm and with a limiting molecular area of 21 A2/amino acid. Under low surface pressure (less than 0.5 dyne/cm), it behaves as a monomer at the interface. It binds reversibly to the surface of phosphatidylcholine-coated triolein particles with a diameter of 26 nm from the aqueous phase in which most of the molecules are tetramerized. An apparent dissociation constant (Kd), 1.2 X 10(-6) M (monomeric molarity) or 40 mg/l, is substantially larger than those of the other apolipoproteins, while a binding saturation level (N), 0.8 amino acid/surface phospholipid, is equivalent to the N values of those proteins (Tajima, S., Yokoyama, S., and Yamamoto, A. (1983) J. Biol. Chem. 258, 10073-10082). Content of alpha-helix increases slightly when it is transferred from the aqueous phase to the lipid surface. The results are consistent with a model that amphiphilic alpha-helical conformation is responsible both for self-association and surface binding and suggest that apo-E may easily dissociate from the lipoprotein surface to form a self-associated soluble tetramer.     

The self-association of human spectrin at high concentration.

The self-association of purified human spectrin has been studied at sedimentation equilibrium over a wide range of concentration (0-20 g/L) at 30 degrees C and pH 7.5. Coincidence of apparent weight average molecular weight and omega (r) plots as a function of total spectrin concentration indicated that equilibrium was attained and that no significant concentration of solute was incapable of participating in the self-association reaction. Under these conditions, no significant dissociation of the heterodimer to component polypeptide chains could be detected. The behavior of spectrin between 0 and 20 g/L can be described reasonably well by a cooperative isodesmic model, in which the protomer for association is the alpha beta heterodimer. With this model, the equilibrium constant for the heterodimer-tetramer step, K24, is 2 x 10(6) M-1, and K(iso), the equilibrium constant describing all other steps, is approximately 0.2 x 10(6) M-1. The returned value of the second virial coefficient for this model, 1.0 x 10(-7) L mol g-2, is consistent with the lower limit of values calculated for the heterodimer from the charge and Stokes radius of spectrin. On the other hand, the attenuated indefinite association model fails to describe the self-association of spectrin adequately over the range 0-20 g/L. Systematic decreases in the estimates of the second virial coefficient and the equilibrium constants for association beyond the tetramer suggest that the assumption of a single value of the second virial coefficient may not be appropriate for spectrin, and that non-ideality would best be taken into account by consideration of the detailed solution composition.     

Self-association of some anthocyanins in neutral aqueous solution

A good contrast in optical properties caused by self-association was found between malvin and cyanin quinonoidal bases. Circular dichroism measurements of the pigments in neutral aqueous solutions show that molecules of the malvin quinonoidal base self-associate quickly and the conformational orientation of the aggregates is opposite to those formed by cyanin quinonoidal bases. Hypsochromism and hypochromism in the visible absorption also occurred on the formation of malvin aggregates. CD comparisons of malvin, hirsutin and 4′-O-methylmalvin suggest that a methoxyl group in the B-ring of the anthocyanidin strongly suppresses self-association. It is proposed that the driving forces for self-association are mainly hydrophobic interactions among the aromatic nuclei stacked parallel to each other which are surrounded by the hydrophilic glucose moieties in a suitable spatial arrangement. Furthermore, the glucose moiety at the 5-position rather than that at the 3-position plays an important role in the self-association of these anthocyanidin 3,5-diglucosides. Addition of sodium chloride promotes self-association and the greater stability of the anthocyanins in solution.     

Light-scattering studies of the alpha-ketoglutarate dehydrogenase complex from escherichia coli. I. Characterization of the self-association of the complex

The self-association of Escherichia coli alpha-ketoglutarate dehydrogenase complex (KGDC) purified by a column Chromatographic technique, was characterized by light-scattering photometry. The complex adopts a solution conformation somewhat larger than that observed in the electron microscope. The evidence suggests a nonideal indefinite self-association model for KGDC in KCl, phosphate buffer. The KGDC monomer has a molecular charge of about -3 x 10(2) at neutral pH. The self-association is promoted by increasing KCl concentrations, pH (in the range from 6.3 to 7.4) and temperature (from 20 to 30 degrees C). The effects of pH changes suggest a release of protons during the self-association and a minor 'preferential' interaction of phosphate ions. For the association of one monomer to the aggregate at neutral pH and 25 degrees C. DeltaG degrees = -7.8 kcal mol(-1). DeltaH degrees = 24 kcal mol(-1) and DeltaS degrees = 1.1 x 10(2) cal mol(-1) K(-1). These data indicate that hydrophobic interactions drive the association. Thermodynamically, the self-association of KGDC is a complex phenomenon and may serve to stabilize the enzyme complex in solution.     

Self-association of lysozyme. Thermochemical measurements: effect of chemical modification of Trp-62, Trp-108, and Glu-35.

Heats of dilution and of saccharide binding for hen egg white lysozyme have been measured at 30 degrees, 0.1 ionic strength, and pH 7 over the range 3 to 95 mg of protein/ml. The concentration dependence of the apparent relative molar enthalpy of lysozyme derived from these results gives the thermodynamic parameters for the formation of an intermolecular contact in an indefinite (head-to-tail) self-association process as delta G 0 = -3.9 kcal/mol, delta H 0 = -6.4 kcal/mol, and delta S 0 = -8,3 e.u. Oxindolealanine-62-lysozyme does not undergo self-association reactions that can be detected calorimetrically. This derivative reacts with native lysozyme to form hybrid polymeric species with free energy and enthalpy of interaction similar to those for the polymers of native lysozyme. These results are consistent with the intermolecular contact in the self-assocaition of lysozyme being asymmetric (head-to-tail). The heat of dilution of the derivative of lysozyme in which Glu-35 is blocked as the ester with oxindolealanine-108 is like that observed for native lysozyme in acid solution and is independent of pH. The concentration difference spectrum that develops through self-association is of the shape expected for introduction of an indole chromophore into a charge-free region of the intermolecular contact. The foregoing results indicate that Glu-35 and Trp-62 are part of the contact, that perturbation of Trp-108 does not make a principle contribution to the concentration difference spectrum, and that no acid group other than Glu-35 is perturbed by self-association. There is a small change in the heat of (GlcNAc)3 binding over the range 0.005 to 0.034 M saccharide. These data give the value of -1 kcal/mol for the enthalpy change for formation of the 2:1 saccharide-enzyme complex (ES2) from ES and S.     

Characterization of HIV-1 p24 self-association using analytical affinity chromatography.

Analytical affinity chromatography (AAC) was used to detect and quantitate the self-association of p24gag, the major structural capsid protein of human immunodeficiency virus (HIV-1). p24gag was immobilized on a hydrophilic polymer (methacrylate) chromatographic support. The resulting affinity column was able to interact with soluble p24, as judged by the chromatographic retardation of the soluble protein upon isocratic elution under nonchaotropic binding conditions. The variation of elution volume with soluble protein concentration fit to a monomer-dimer model for self-association. The soluble p24-immobilized p24 association process was observed using both frontal and zonal elution AAC at varying pH values; the dissociation constant was 3-4 x 10(-5) M at pH 7. That p24 monomer associates to dimers was determined in solution using analytical ultracentrifugation. The solution Kd was 1.3 x 10(-5) M at pH 7. AAC in the zonal elution mode provides a simple and rapid means to screen for other HIV-1 macromolecules that may interact with p24 as well as for modulators, including antagonists, of HIV p24 protein assembly.     

Estimation of direct-contact fraction for phenanthrene in surfactant solutions by toxicity measurement

The toxicity of solutions containing nonionic surfactants Tween 80, Brij 35 and/or phenanthrene to Pseudomonas putida ATCC 17484 was investigated. The fraction of direct contact between micellar-phase phenanthrene and bacterial cell surface was estimated by using the toxicity data and a mathematical model. The mathematical model was used to calculate phenanthrene concentration in the micellar phase and aqueous pseudophase separately. The first-order death rate constant increased from 0.088+/-0.016 to 0.25+/-0.067 h(-1) when the phenanthrene concentration was increased from 0 to 5.17 x 10(-6)M (equals water solubility). The intrinsic toxicity of surfactant was higher in Brij 35 than in Tween 80. When phenanthrene concentration was increased to 9.7 x 10(-5)M in surfactant solutions, the death rate constant increased to 1.8 +/- 0.024 and 0.41 +/- 0.088 h(-1) for 8.4 x 10(-4)M Brij 35 and 7.6 x 10(-4)M Tween 80. The direct-contact fraction was 0.083 and 0.044 for Brij 35 and Tween 80, respectively, under these conditions using exponential model. The toxicity increased with increasing phenanthrene concentration at a fixed surfactant concentration. The toxicity decreased with increasing the surfactant concentration at a fixed phenanthrene concentration due to decreased contact of bacteria with phenanthrene present in the interior of surfactant micelles.     

Ionic strength induced structure in histone H4 and its fragments.

The salt-induced folding and self-association of histone H4 and its fragments (1-23), (25-67), (69-84), and (69-102) have been studied at the same molar concentration (1 mM) by nuclear magnetic resonance (NMR), circular dichroism (CD), and ir spectroscopy. Byeach of these techniques intact histone H4 exhibited a fast structural change, involving the formation of alphaR helix and aggregation, and also a slow change involving beta-structure formation. Fragment (25-67) was found to behave in a manner similar to the intact molecule for the fast change, showing both helix formation and aggregation but exhibited no time-dependent effects. All the other fragments were found to be essentially noninteracting. It is concluded that (25-67) contains the rgion critical for the folding and self-association of histone H4. On the basis of these results a model is proposed for the self-association of histone H4 in which helix is located between residues 49 and 73, while the beta structure lies between 74 and the C-terminus.     

Solution effects on the self-association of a water-soluble peptoid

A desire to replicate the structural and functional complexity of proteins with structured, sequence-specific oligomers motivates study of the structural features of water-soluble peptoids (N-substituted glycine oligomers). Understanding the molecular-level details of peptoid self-assembly in water is essential to advance peptoids' application as novel materials. Peptoid 1 , an amphiphilic, putatively helical peptoid previously studied in our laboratory, shows evidence of self-association in aqueous solution. In this work, we evaluate how changes to aqueous solution conditions influence the self-association of 1 . We report that changes to pH influence the fluorescence and CD spectroscopic features as well as the peptoid's interaction with a solvatochromic fluorophore and its apparent size as estimated by size exclusion chromatography. Addition of guanidine hydrochloride and ammonium sulfate also modulate spectroscopic features of the peptoid, its interaction with a solvatochromic fluorophore, and its elution in size exclusion chromatography. These data suggest that the ordering of the self-assembly changes in response to pH and with solvent additives and is more ordered at higher pH and in the presence of guanidine hydrochloride. The deeper understanding of the self-association of 1 afforded by these studies informs the design of new stimuli-responsive peptoids with stable tertiary or quaternary structures.     

Diversity in the supramolecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole with modified cyclodextrins: Implications for physicochemical properties and antiparasitic activity

The molecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole (G2), an antiprotozoa with poor aqueous solubility, with 2-hydroxypropyl-α-cyclodextrin (HPαCD), methyl-β-cyclodextrin (MβCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) were examined. The aqueous solubility enhancement by cyclodextrins (CDs) was evidenced in phase-solubility diagrams, and the stoichiometry of G2/CD systems was determined by Job's plots. Two-dimensional NMR spectroscopic data revealed that a different mode of interaction took place between G2 and CDs in solution. With HPαCD, a non-inclusion complex was generated. In the case of MβCD, a typical host-guest system was obtained and with HPβCD a partial inclusion complex through the narrow side of the macrocycle was formed. ESI-mass spectrometric data confirmed the stoichiometry and mode of interaction of these systems in solution. Solid-state characterization (scanning calorimetry and powder X-ray diffraction) supported the inclusion complex formation. The leishmanicidal activity, trypanocidal activity and non-toxic profile of G2/MβCD showed the advantages of using this inclusion complex to promote the biological assays extension of G2.     

Temperature-dependent beta-sheet formation in beta-amyloid Abeta(1-40) peptide in water: uncoupling beta-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimer's beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.     

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism.

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)