Light scattering studies of the binding of bovine serum albumin to a cationic polyelectrolyte

Poly(dimethyldiallylammonium chloride) (PDMDAAC) exhibits a strong electrostatic interaction with bovine serum albumin (BSA) at pH 8.0 in 0.16M NaCl. Electrophoretic, dynamic, and static light scattering suggest that the mode of binding of BSA to PDMDAAC depends upon the weight concentration ratio (r) of BSA to PDMDAAC. When r is smaller than ca. 10, the system exhibits characteristics of cooperative binding, in that the BSA molecules are inhomogeneously distributed among the polymer chains, and free PDMDAAC molecules coexist with complex. When r reaches ca. 10, the amount of free PDMDAAC is too small to be observed. Further increase in r leads to a secondary binding process along with an increase in the amount of free protein. Hydrophobic interactions among the bound BSA are proposed as the driving force for the cooperative binding. © 1996 John Wiley & Sons, Inc.     

Influence of the NaCl or CaCl2 concentration on the structure of heat-set bovine serum albumin gels at pH 7

The structure of heat-set systems of the globular protein bovine serum albumin (BSA) was investigated at pH 7 in different salt conditions (NaCl or CaCl(2)) using light scattering. Cross-correlation dynamic light scattering was used to correct for multiple scattering from turbid samples. After heat treatment, aggregates are formed whose size increases as the protein concentration increases. Beyond a critical concentration that decreases with increasing salt concentration, gels are formed. The heterogeneity and the reduced turbidity of the gels were found to increase with increasing salt concentration and to decrease with increasing protein concentration. The structure of the gels is determined by the strength of the repulsive electrostatic interactions between the aggregated proteins. The results obtained in NaCl are similar to those reported in previous studies for other globular proteins. CaCl(2) was found to be much more efficient in reducing electrostatic interactions than NaCl at the same ionic strength.     

Interactions of urocanic acid with bovine serum albumin and the influence of pH on binding affinities: a docking simulation study

In the present study, the interactions of urocanic acid (UA) with bovine serum albumins (BSA) at pH 5.0 and 7.4 were investigated by means of docking simulations. The binding modes of trans- and cis-UA to BSA at pH 5.0 and 7.4 were analysed. In addition, the theoretically predicted binding abilities of zwitterion and anion of UA with BSA are in good agreement with the experimental results. Through comparison with the binding patterns, we revealed that the stronger interactions of UA anion with BSA relative to the zwitterion primarily result from: (1) the increased number of hydrogen bonds between UA anion and BSA; (2) the attractive electrostatic interaction between the deprotoned carboxyl group in UA anion and Arg433 in comparison with the repulsion between the imidazole moiety in zwitterion and the same residue in BSA. This provides a rational explanation for the experimental finding that the binding of UA to BSA at pH 7.4 is much stronger than at pH 5.0.     

How does dextran sulfate prevent heat induced aggregation of protein? The mechanism and its limitation as aggregation inhibitor

The effect of dextran sulfate on protein aggregation was investigated to provide the clues of its biochemical mechanism. The interaction between dextran sulfate and BSA varied with the pH values of the solution, which led to the different extent of aggregation prevention by dextran sulfate. Light scattering data with thermal scan showed that dextran sulfate suppressed BSA aggregation at pH 5.1 and pH 6.2, while it had no effect at pH 7.5. Isothermal titration calorimetric analysis suggested that the pH dependency of the role of dextran sulfate on BSA aggregation would be related to the difference in the mode of BSA-dextran sulfate complex formation. Isothermal titration calorimetric analysis at pH 6.2 indicated that dextran sulfate did not bind to native BSA at this pH, but interacted with partially unfolded BSA. While stabilizing native form of protein by the complex formation has been suggested as the suitable mechanism of preventing aggregation, our observation of conformational changes by circular dichroism spectroscopy showed that strong electrostatic interaction between dextran sulfate and BSA rather facilitated the denaturation of BSA. Combining the data from isothermal titration calorimetry, circular dichroism, and dynamic light scattering, we found that the complex formation of the intermediate state of denatured BSA with dextran sulfate is a prerequisite to suppress the aggregation by preventing further oligomerization/aggregation process of denatured protein.     

Quantitative characterization of the concentration-dependent interaction between molecules of Dextran 70 in aqueous solution: Measurement and analysis in the context of thermodynamic and compressible sphere models

The static light scattering and sedimentation equilibrium of solutions of Dextran 70 were measured as functions of concentration up to 100 g/L in pH 7.4 phosphate-buffered saline at temperatures between 5 and 37 °C. The concentration dependence of scattering intensity and the apparent molar mass obtained from sedimentation equilibrium were found to be nearly independent of temperature over this range to within the uncertainty of measurement. Global analysis of the concentration dependence of both properties yielded a reliable estimate of the concentration-dependent thermodynamic activity coefficient, a quantitative measure of the free energy of self-interaction. The self-interaction between Dextran molecules is compared with that of a globular protein (BSA) and a highly crosslinked polymer of similar molar mass (Ficoll 70). The observed concentration dependence of the free energy of Dextran self-interaction may be quantitatively accounted for by a semi-empirical model in which the polymer molecule is represented by a compressible sphere.     

The Importance of Protein-Protein Interactions on the pH-Induced Conformational Changes of Bovine Serum Albumin: A Small-Angle X-Ray Scattering Study

The combined effects of concentration and pH on the conformational states of bovine serum albumin (BSA) are investigated by small-angle x-ray scattering. Serum albumins, at physiological conditions, are found at concentrations of ∼35-45 mg/mL (42 mg/mL in the case of humans). In this work, BSA at three different concentrations (10, 25, and 50 mg/mL) and pH values (2.0-9.0) have been studied. Data were analyzed by means of the Global Fitting procedure, with the protein form factor calculated from human serum albumin (HSA) crystallographic structure and the interference function described, considering repulsive and attractive interaction potentials within a random phase approximation. Small-angle x-ray scattering data show that BSA maintains its native state from pH 4.0 up to 9.0 at all investigated concentrations. A pH-dependence of the absolute net protein charge is shown and the charge number per BSA is quantified to 10(2), 8(1), 13(2), 20(2), and 26(2) for pH values 4.0, 5.4, 7.0, 8.0, and 9.0, respectively. The attractive potential diminishes as BSA concentration increases. The coexistence of monomers and dimers is observed at 50 mg/mL and pH 5.4, near the BSA isoelectric point. Samples at pH 2.0 show a different behavior, because BSA overall shape changes as a function of concentration. At 10 mg/mL, BSA is partially unfolded and a strong repulsive protein-protein interaction occurs due to the high amount of exposed charge. At 25 and 50 mg/mL, BSA undergoes some re-folding, which likely results in a molten-globule state. This work concludes by confirming that the protein concentration plays an important role on the pH-unfolded BSA state, due to a delicate compromise between interaction forces and crowding effects.     

Further studies on the contribution of electrostatic and hydrophobic interactions to protein adsorption on dye-ligand adsorbents.

The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption.     

Adsorption of BSA on strongly basic chitosan: Equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a new adsorbent, a strongly basic crosslinked chitosan (Chitopearl 2503), which is hard and is not compressed by pressure in a column, have been presented and compared with diethylaminoethyl (DEAE) Sepharose Fast Flow (hard gel). In Chitopearl 2503, when only buffer existed in the BSA solution, the isotherm was not affected by the initial concentration of BSA but it was affected by pH considerably. The isotherm was favorable when pH >/= pl ( congruent with 4.8). When NaCl existed in the BSA solution, the amount of BSA absorbed on the resin decreased with increasing concentration of NaCl. When the concentration of NaCl was 200 mol/m(3), the resin did not adsorb BSA at all. The equilibrium data were correlated by the Langmuir equation reasonably well. The BSA may be adsorbed mainly by electrostatic attraction between negatively charged BSA and positively charged quanternary ammonium groups at pH > pl and by protonation reaction of the primary ammonium groups by weak acid groups of BSA at pH = pl. These are confirmed by measuring the amount of inorganic ion exchanged for BSA. In DEAE Sepharose Fast Flow, the isotherm was favorable when pH > pl but unfavorable ar pH = pl. The saturation capacity of BSA on Chitopearl 2503 is about 1.3 to 2.2 times larger than that on DEAE Sepharose Fast Flow. (c) 1994 John Wiley & Sons, Inc.     

A New Preparation Method of Coenzyme A with Microorganisms

The structure of aggregates formed by heating dilute BSA solution was analyzed with the fractal concept using light scattering methods. BSA was dissolved in HEPES buffer of pH 7.0 and acetate buffer of pH 5.1 to 0.1% and 0.001% solutions, respectively, and heated at 95°C, varying the heating time t_a. The fractal dimension D_f of the aggregate in the solution was evaluated from static light scattering experiments. The polydispersity exponent τ and the average hydrodynamic radius <R_h> of the aggregates were calculated from dynamic light scattering experiments using master curves obtained by Klein et al. The values of D_f and τ of heat-induced aggregates of BSA at pH 7.0 were about 2.1 and 1.5, respectively, the values of which agreed with those predicted by the reaction-limited cluster–cluster aggregation (RLCCA) model. On the other hand, D_f of heat-induced aggregates at pH 5.1 was about 1.8, which agreed with that predicted by the diffusion-limited cluster–cluster aggregation (DLCCA) model. The dependence of <R_h> for the sample of pH 7.0 on t_a was similar to that of the polystyrene colloids reported previously.     

Static Light Scattering From Concentrated Protein Solutions II: Experimental Test of Theory for Protein Mixtures and Weakly Self-Associating Proteins

Using an experimental technique recently developed in this laboratory (Fernández C. and A. P. Minton. 2008. Anal. Biochem. 381:254-257), the Rayleigh light scattering of solutions of bovine serum albumin, hen egg white ovalbumin, hen egg white ovomucoid, and binary mixtures of these three proteins was measured as a function of concentration at concentrations up to 125 g/L. The measured concentration dependence of scattering of both pure proteins and binary mixtures is accounted for nearly quantitatively by an effective hard particle model (Minton A. P. 2007. Biophys. J. 93:1321-1328) in which each protein species is represented by an equivalent hard sphere, the size of which is determined by the nature of repulsive interactions between like molecules under a given set of experimental conditions. The light scattering of solutions of chymotrypsin A was measured as a function of concentration at concentrations up to 70 g/L at pH 4.1, 5.4, and 7.2. At each pH, the measured concentration dependence is accounted for quantitatively by an effective hard particle model, according to which monomeric protein may self-associate to form an equilibrium dimer and, depending upon pH, an equilibrium pentamer or hexamer.     

Biothermodynamic analysis of BSA adsorption to alum gel using isothermal titration calorimetry

In this study, we used ITC (isothermal titration calorimetry) to quantitatively investigate the impacts of temperature and protein concentration on adsorption behavior on a solid surface, using BSA (bovine serum albumin) as a model protein, and alum (aluminum hydroxide) gel as an adsorbent. The zeta potential measurement for alum gel (0.25 mV at pH 9.3) revealed that its surface charge was not strong enough for electrostatic interaction. ITC analysis showed that the BSA-alum gel interaction was entropy-driven, suggesting that during adsorption, water molecules were expelled from the hydration layers of the alum gel and BSA. Therefore, the major mechanism for the BSA-alum gel interaction was hydrophobic interaction rather than electrostatic interaction. This biothermodynamic approach can be helpful not only to identify interaction mechanisms, but also to explore the optimum conditions for protein-adsorbent interactions.     

Dermatan sulfate as a stabilizer for protein stability in poly(lactide-co-glycolide) depot

Dermatan sulfate (DS), a glycosaminoglycan family, was investigated as a additive to enhance the stability of therapeutic protein with low p/ value loaded in poly(lactide-co-glycolide) (PLGA) microspheres prepared by water-in-oil-in-water (W₁/O/W₂) method. DS maintains negative charge below pH 3.0 because of its sulfate groups, while most anionic polymer with carboxyl groups becomes neutral charge at that pH. Thus, at pH 3.0 DS can form a polyelectrolyte complex with a protein with lower p/ such as exendin-4, insulin, and human growth hormone. In order to complex with DS, bovine serum albumin (BSA) was employed as a model protein, which has low p/value (p/= 4.8). The complex prepared at pH 3.0 showed a nano-size in the range of 100∼200 nm with a mono distribution. During the preparation of PLGA depot, DS concentration in water phase increases with decreasing the formation of non-covalent BSA aggregates and enhancing BSA loading efficiency. It means that DS/BSA complex system enabled to keep a stability of BSA at the water/organic interface. In an in vitro BSA release test, PLGA depot with DS exhibited a lower initial burst kinetic than only PLGA depot and continuous BSA release in almost 100% for 23 days. From the results, it was concluded that DS as an additive in PLGA depot, has a potential for the long-term delivery of therapeutic proteins with lower p/ value.     

Study on the interaction between pelargonidin‐3‐O‐glucoside and bovine serum albumin using spectroscopic,transmission electron microscopy and molecular modeling techniques

The aim of this study is to evaluate the binding behavior between pelargonidin‐3‐O‐glucoside (P3G) and bovine serum albumin (BSA) using multi‐spectroscopic, transmission electron microscopy (TEM) and molecular docking methods under physiological conditions. Fluorescence spectroscopy and time‐resolved fluorescence showed that the fluorescence of BSA could be quenched remarkably by P3G via a static quenching mechanism, and there is a single class of binding site on BSA. In addition, the thermodynamic functions ΔH and ΔS were –21.69 kJ/mol and 24.46 J/mol/K, indicating that an electrostatic interaction was a main acting force. The distance between BSA and P3G was 2.74 nm according to Förster's theory, illustrating that energy transfer occurred. In addition, the secondary structure of BSA changed with a decrease in the α‐helix content from 66.2% to 64.0% as seen using synchronous fluorescence, UV/vis, circular dichroism and Fourier transform infrared spectroscopies, whereas TEM images showed that P3G led to BSA aggregation and fibrillation. Furthermore, site marker competitive experiments and molecular docking indicated that P3G could bind with subdomain IIA of BSA. The calculated results of the equilibrium fraction showed that the concentration of free P3G in plasma was high enough to be stored and transported from the circulatory system to its target sites to provide therapeutic effects. Copyright © 2015 John Wiley & Sons, Ltd.     

The hyaluronan–protein complexes at low ionic strength: How the hyaluronidase activity is controlled by the bovine serum albumin

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at low HAase over HA concentration ratio and under low ionic strength conditions. The reason is the ability of long HA chains to form electrostatic and non-catalytic complexes with HAase. For a given HA concentration, low HAase concentrations lead to very low hydrolysis rates because all the HAase molecules are sequestered by HA, whilst high HAase concentrations lead to high hydrolysis rates because the excess of HAase molecules remains free and active. At pH 4, non-catalytic proteins like bovine serum albumin (BSA) are able to compete with HAase to form electrostatic complexes with HA, liberating HAase which recovers its catalytic activity. The general scheme for the BSA-dependency is thus characterised by four domains delimited by three noticeable points corresponding to constant BSA over HA concentration ratios. The existence of HA–protein complexes explains the atypical kinetic behaviour of the HA / HAase system. We also show that HAase recovers the Michaelis–Menten type behaviour when the HA molecule complexed with BSA in a constant complexion state, i.e. with the same BSA over HA ratio, is considered for substrate. When the ternary HA / HAase / BSA system is concerned, the stoichiometries of the HA–HAase and HA–BSA complexes are close to 10 protein molecules per HA molecule for a native HA of 1 MDa molar mass. Finally, we show that the behaviour of the system is similar at pH 5.25, although the efficiency of BSA is less.     

Activity coefficients of salts in highly concentrated protein solutions: I. Alkali chlorides in isoionic bovine serum albumin solutions

In order to understand the thermodynamic state of simple salts in living cells, the mean activity coefficients of LiCl, NaCl, KC1, RbCl, CsCl were determined in concentrated isoionic bovine serum albumin (BSA) solutions by use of the EMF method with ion exchange membrane electrodes. The protein concentration range extended up to 22 wt %, whereas the salt concentration was kept constant at 0.1 mole per kilogram water. These solutions may be regarded as crude but appropriate model systems for the cytoplasm of cells as far as type and magnitude of the macromolecular component influence on the chemical potential of the salts is concerned. The mean stoichiometric activity coefficients of the alkali chlorides in the isoionic BSA solutions decreased linearly with the protein molality; this decrease, however, did not exceed ca. 10% compared with the pure 0.1 molal salt solutions. Only very small differences in the behaviour of the different alkali chlorides were observed. The results may be interpreted by the superposition of the effects of specific Cl^? ion binding to BSA and BSA bound “non-solvent” water with probably electrostatic long range interactions of the BSA(Cl^?)_v polyions with the salt ions in solution. The resulting mean activity coefficients, corrected for ion binding and non-solvent water, showed a very slight linear dependence on the protein concentration. The departure from the value in the pure 0.1 molal salt solutions did not exceed ± 2%.     

Influence of pH and ionic strength on the steric mass-action model parameters around the isoelectric point of protein

The ion-exchange equilibrium and the dependence of the parameters in the steric mass-action (SMA) model on salt concentration and buffer pH around the isoelectric point of protein were studied. Bovine serum albumin (BSA, isoelectric point = 5.4) was used as a model protein and DEAE Sepharose FF as an ion exchanger. Finite batch adsorption experiments and isocratic elution chromatography were performed for the determination of the model parameters (i.e., characteristic charge, equilibrium constant, and steric factor). The results showed that pH had significant effects on the parameters. With an increase of pH from 4.5 to 6.5, the characteristic charge increased from 0.9 to 3.0 and leveled off as a plateau at pH above 5.5. The charge groups in the contact region of protein surface were considered to play a crucial role on the characteristic charge. The decrease of pH and increase of salt concentration lowered the absolute value of the zeta potential of the protein surface and led to a decrease of the equilibrium constant. The steric factor remained unchanged at about 31 at pH 5.5 and 6.0 and increased to 44.5 at pH 5.0 and 96.8 at pH 4.5, mainly as a result of the lower adsorption capacity of BSA at pH <5.5. Furthermore, the increase of the molecular volume of BSA at pH 4.5 would be an additional reason for the increase of the steric factor. Taking into account the effect of the pH and salt concentration on these parameters, the SMA model described the ion exchange equilibrium of protein more accurately.     

Deciphering binding mechanism between bovine serum albumin and new pyrazoline compound K4

The binding mechanism of a new and possible drug candidate pyrazoline derivative compound K4 and bovine serum albumin (BSA) was investigated in buffer solution (pH 7.4) using ultraviolet–visible light absorption and steady‐state and synchronous fluorescence techniques. The fluorescence intensity of BSA was quenched in the presence of K4 . The quenching process between BSA and K4 was examined at four different temperatures. Decrease of the quenching constants calculated using the Stern–Volmer equation and at increasing temperature suggested that the interaction BSA– K4 was realized through a static quenching mechanism. Synchronous fluorescence measurements suggested that K4 bounded to BSA at the tryptophan region. Fourier transform infrared spectroscopy results showed that there was no significant change in polarity around the tryptophan residue The forces responsible for the BSA– K4 interaction were examined using thermodynamic parameters. In this study, the calculated negative value of ΔG, the negative value of ΔH and the positive value of ΔS pointed to the interaction being through spontaneous and electrostatic interactions that were dominant for our cases. This study provides a very useful in vitro model to researchers by mimicking in vivo conditions to estimate interactions between a possible drug candidate or a drug and body proteins.     

Adsorption of BSA on QAE-dextran: equilibria

Equilibrium isotherms for adsorption of bovine serum albumin (BSA) on a strong-base (QAE) dextran-type ion exchanger have been determined experimentally. They were not affected by the initial concentration of BSA but were affected by pH considerably. They were correlated by the Langmuir equation when pH >/= 5.05 and by the Freundlich equation of pH 4.8, which is close to pl approximately 4.8 of BSA. The contribution of ion exchange to adsorption of BSA on the ion exchanger was determined experimentally. The maximum amounts of inorganic anion exchanged for BSA were 1% and 0.4% of the exchange capacity of the ion exchanger at pH 6.9, respectively. Since the effect of the ion exchange on the adsorption appeared small, BSA may be adsorbed mainly by electrostatic attraction when pH >/= 5.05 and by hydrophobic interaction or hydrogen bonding at pH 4.8. When NaCl coexisted in the solution, the shape of the isotherm was similar to the Langmuir isotherm, but it is shifted to the right. When the concentration of NaCl was 0.2 mol/dm(3), BsA was not adsorbed on the resin. When BSA was dissolved in pure water, the saturation capacity of BSA on HPO(4) (2-),-orm resin was about 2 times larger than that for adsorption from the solution with buffer (pH 6.9 and 8.79). The saturation capacity for adsorption of BSA in pure water on HPO(4) (2-) + H(2)O(4) (-)-from resin was much smaller than that from the solution with buffer. The isotherms for univalent Cl(-)-and H(2)PO(4) (-)-form resin was peculiar; that is, the amount of BSA adsorbed decreased with increasing the liquid-phase equilibrium concentration of BSA. (c) 1993 John Wiley & Sons, Inc.     

pH effects on the hyaluronan hydrolysis catalysed by hyaluronidase in the presence of proteins: Part I. Dual aspect of the pH-dependence

Hyaluronan (HA) hydrolysis catalysed by hyaluronidase (HAase) is strongly inhibited when performed at a low ratio of HAase to HA concentrations and at low ionic strength. This is because long HA chains can form non-active complexes with HAase. Bovine serum albumin (BSA) is able to compete with HAase to form electrostatic complexes with HA so freeing HAase which then recovers its catalytic activity. This BSA-dependence is characterised by two main domains separated by the optimal BSA concentration: below this concentration the HAase activity increases when the BSA concentration is increased, above this concentration the HAase activity decreases. This occurs provided that HA is negatively charged and BSA is positively charged, i.e. in a pH range from 3 to 5.25. The higher the pH value the higher the optimal BSA concentration. Other proteins can also modulate HAase activity. Lysozyme, which has a pI higher than that of BSA, is also able to compete with HAase to form electrostatic complexes with HA and liberate HAase. This occurs over a wider pH range that extends from 3 to 9. These results mean that HAase can form complexes with HA and recover its enzymatic activity at pH as high as 9, consistent with HAase having either a high pI value or positively charged patches on its surface at high pH. Finally, the pH-dependence of HAase activity, which results from the influence of pH on both the intrinsic HAase activity and the formation of complexes between HAase and HA, shows a maximum at pH 4 and a significant activity up to pH 9.     

Effects of pH and ionic strength on the structure of collagen fibrils

The roles of pH and ionic strength on the structure and stability of collagen fibrils have been investigated by means of x-ray and neutron diffraction techniques. High-angle x-ray diffraction shows that a salt concentration of 0.5M KCl is sufficient to reduce the osmotic swelling and related disordering in the pH range 1–3. The relative intensities of the low-angle meridional x-ray and neutron diffraction Bragg reflections vary with pH. Difference Fourier syntheses between pH 7 and 1.6 data indicate, for both x-ray and neutron diffraction, a reduced scattering contribution from the telopeptides at low pH. Lyotropic relaxation is a crucial step in the appearance at low pH of a doubling of the 668-Å axial periodicity (D) of collagen fibrils. These results suggest that electrostatic interactions are essential for the structural stability of the telopeptide regions and of the 1D and 3D intermolecular staggers between collagen molecules.