N-NBD-L-alpha-dilauroylphosphatidylethanolamine. A new fluorescent probe to study spontaneous lipid transfer.

Migration of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-L-alpha-dilauroylphosphati dylethanolamine between small sonicated egg phosphatidylcholine vesicles was studied by use of the fluorescence resonance energy transfer method. Contrary to the results of lipid transfer experiments reported for acyl chain NBD-labeled phospholipids (Nichols, J.W. and Pagano, R.E. (1982) Biochemistry 21, 1720-1728), the migration kinetics of N-NBD-DLPE had to be described by a sum of two exponential functions. The fast component (t1/2 approximately equal to 38 min) was assigned to lipid transfer via soluble monomers and the slow component (t1/2 approximately equal to 400 min) to transbilayer motion. A reversible four-stage process is suggested as a kinetic model. Mathematical treatment of this scheme is given yielding an analytical expression for the time dependence of NBD emission intensity. The use of N-NBD-DLPE in the resonance energy transfer measurements offers the advantage of simple chemical synthesis of the fluorescent probe and leads to additional information on transbilayer motion which was not available with the NBD-labeled lipids used so far.

Low pH fusion of mouse liver nuclei with liposomes bearing covalently bound lysozyme.

Lysozyme covalently bound to liposomes induces the fusion of liposomes with isolated mouse liver nuclei. The fusion behavior is very similar to the case of erythrocyte ghosts (Arvinte, T., Hildenbrand, K., Wahl, P. and Nicolau, C. (1986) Proc. Natl. Acad. Sci. USA 83, 962-966). Kinetic studies showed that membrane lipid mixing was completed within 15 min, as indicated from the resonance energy transfer (RET) measurements. For the resonance energy transfer kinetic measurements the liposomes contained L-alpha-dipalmitoylphosphatidylethanolamine (DPPE), labeled at the free amino group with the energy donor 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) or with the energy acceptor tetramethylrhodamine. The lipid mixing at equilibrium was studied by the fluorescence recovery after photobleaching technique (FRAP). Liposomes (with/without lysozyme) containing Rh-labeled DPPE in their membranes were incubated with nuclei at 37 degrees C, pH 5.2, for 30 min. After washing of nuclei by three centrifugations, 60-70% of the initial amount of labeled DPPE was associated with the nuclei in the case of liposomes bearing lysozyme and only 7-10% in the case of liposomes without lysozyme. For the nuclei incubated with liposomes having lysozyme, about 70% of the total Rh-labeled lipids present in the nuclei diffused in the nuclear membrane(s) (lateral diffusion constant of D = (1.4 +/- 0.5) X 10(-9) cm2/s). By encapsulating fluorescein isothiocyanate-labeled dextran of 150 kDa molecular mass into the liposomes and using a microfluorimetric method, it was shown that after the fusion a part of the liposome contents is found in the nuclei interior. In this lysozyme-induced fusion process between liposomes and nuclei or erythrocyte ghosts, the binding of lysozyme to the glycoconjugates contained in the biomembranes at acidic pH seems to be the determining step which explains the high fusogenic property of the liposomes bearing lysozyme.

Low-pH association of proteins with the membranes of intact red blood cells. I. Exogenous glycophorin and the CD4 molecule.

Glycophorin and CD4 proteins are tightly associated with intact human erythrocyte membranes after a short-time incubation at low pH (1-2 min, pH lower than 5, 37 degrees C). Flow cytometry and epifluorescence microscope observations showed that after incubation of red cells with fluorescein isothiocyanate (FITC) labeled glycophorin at pH values lower than 5, the erythrocyte membrane and subsequently formed ghost membranes were fluorescent. Unlabeled glycophorin was reacted with mouse erythrocytes using the same low-pH conditions. Flow cytometry and fluorescence microscopy showed that anti-glycophorin monoclonal antibodies were able to recognize the epitopes of glycophorin associated with the mouse erythrocytes. Kinetic experiments showed that the interaction of FITC-glycophorin with red cell membranes can be monitored by a decrease in the fluorescence intensity. Erythrocyte associated glycophorin was not removed from the membranes after 24 h incubation in human plasma (in vitro, 39 degrees C). A glycoprotein extract containing CD4 was isolated from a T4-lymphoma cell line (CEM). This protein extract was incubated with erythrocytes using the same low-pH conditions. Fluorescently labeled monoclonal antibodies against CD4 stained the red cells after association of CD4 with the membranes. Electron microscopy showed 10 nm immunoglobulin G-coated gold beads associated with CD4-bearing erythrocyte membranes after incubation with anti-CD4 antibodies and then with the gold beads. The potential use of the CD4-erythrocyte complex as a therapeutical agent against acquired immune deficiency syndrome (AIDS) is suggested.

Low-pH association of proteins with the membranes of intact red blood cells. II. Studies of the mechanism.

The low-pH interaction of proteins with erythrocyte membranes has been found to be correlated with pH-induced changes in the erythrocyte membrane. Using a 90 degree lightscattering method it was shown that red blood cell hemolysis was slow between pH 5.8 and 5 (t1/2 above 1 h) but became fast at and below pH 4.7 (t1/2 less than 20 min). At pH 4.7, the presence of glycophorin in the incubation medium inhibited the hemolysis of erythrocytes and this protective effect was found to be dependent on the glycophorin concentration. Electron microscope experiments showed the presence of membrane defects after 10 s incubation at pH 4.6 in the absence of glycophorin in the incubation medium. These defects could further develop into openings with average widths of 14 nm after 1.5 min incubation under the acidic conditions. Fluorescence and flow cytometry studies showed that at pH 4.7, but not at pH 7.4, glycophorin tightly associates with phosphatidylcholine liposomes, and that liposome associated glycophorin molecules are recognized by anti-glycophorin monoclonal antibodies.

Watching amyloid fibrils grow by time-lapse atomic force microscopy.

Late-onset diabetes is typically associated with amyloid deposits of fibrillar amylin in the pancreatic islets. Aqueous synthetic human amylin spontaneously forms polymorphic fibrils in vitro, and this system was used to examine the dynamics of fibril assembly. By time-lapse atomic force microscopy (AFM), the growth of individual amylin fibrils on a mica surface was observed over several hours. Prominent was the assembly of a protofibril with an elongation rate in these experiments of 1.1(+/-0.5) nm/minute. The assembly of higher order polymorphic fibrils was also observed. Growth of the protofibrils was bidirectional, i.e. it occurred by elongation at both ends. This ability of AFM to continuously monitor growth, directionality, and changes in morphology for individual fibrils, provides a significant advantage over spectroscopy-based bulk methods which average the growth of many fibrils and typically require 100 to 1000-fold more protein. The time-lapse AFM procedure used for human amylin here is thus likely to be applicable to fibril formation from other amyloid proteins and peptides.

Modulation of antigenicity related to changes in antibody flexibility upon lyophilization.

Lyophilization is frequently used to increase the shelf-life of biopharmaceuticals containing antibodies. A case in which an anti-idiotypic antibody, MMA 383, substantially lost its in vivo immunogenic properties although the protein was not degraded, is investigated. The scanning transmission electron microscope allowed the MMA 383 Fab and Fc moieties to be resolved. By averaging the single antibodies, the angle between the Fab moieties can be calculated. Non-lyophilized antibodies displayed a wider range of shapes than their reconstituted, lyophilized counterparts. Accordingly, the angle between the two Fab fragments varied more, indicating greater flexibility. The tryptophan steady-state fluorescence intensity, steady-state fluorescence anisotropy and fluorescence lifetime, were smaller for the lyophilized antibodies. These were also more resistant towards thermal denaturation/aggregation. Circular dichroism spectra detected temperature-dependent differences between the two antibody types in the 236 nm region. The subtle but reproducible structural changes induced by lyophilization may be related to the loss of in vivo immunogenic properties.

Dextran protection of erythrocytes from low-pH-induced hemolysis.

Low-pH-induced hemolysis of erythrocytes is inhibited by dextrans. The protective effect was observed with dextrans larger than 40 kDa. Electron microscopy showed dextrans of 150 kDa in a tight association with the erythrocyte membrane. These results indicate that dextrans stop the low-pH-induced hemolysis by interacting with the acid-induced defects in the erythrocyte membrane [(1989) Biochim. Biophys. Acta, in press].

Red blood cells bearing CD4 bind to gp120 covered plates and aggregate with cells expressing gp120.

Full-length recombinant CD4 (rCD4) was bound to human red blood cell membranes using a low-pH incubation procedure. The red blood cells bearing recombinant CD4 (RBC-rCD4+) specifically bound to gp 120-covered plates. The binding showed a threshold effect at low rCD4 concentrations per RBC. The binding of RBC-rCD4+ to gp120 plates was not inhibited by plasma proteins (incubation in human blood plasma). Preincubation of the RBC-rCD4+ with monoclonal anti-CD4 antibodies blocked the binding to the gp120-covered plates. Incubation of chinese hamster ovary (CHO) fibroblasts, which constitutively express the HIV envelope protein gp120 with RBC-rCD4+, resulted in the formation of large cell aggregates. These in vitro data suggest that RBC-rCD4+ retain affinity for gp120 and thus could have potential therapeutical value in AIDS treatment.

Transforming growth factor beta-3 crystals as reservoirs for slow release of active TGF-beta3.

Transforming growth factor betas (TGF-betas) play critical roles in many diseased states and injury repair processes. Exogenous delivery of TGF-beta may thus have therapeutic applications. Here, crystals of TGF-beta3 (TGF-beta3) are being evaluated as protected reservoirs for sustained local release. A sensitive Mv1Lu cell growth inhibition assay established that in vitro, active TGF-beta3 can be delivered from physically stable crystals. Non-sink release experiments revealed that crystal solubility at pH 7.4 was higher in cell culture medium (2.7+/-0.1 microg/ml) than in saline buffers (approximately 1-1.5 microg/ml, P<0.05). Addition of serum induced a five-fold delay in equilibration of soluble-crystal TGF-beta3. Semi-sink experiments cumulated in higher TGF-beta3 release than under non-sink conditions; the observed steady states correlated with crystal solubility and the frequency of buffer exchange. Release of TGF-beta3 from crystals was also strongly dependent on solubility changes as affected by pH. At neutral pH the solubilities were the lowest, and increased with both higher and lower pH. The results indicate that TGF-beta3 crystals may have promising features for local pH-triggered sustained-release applications.

Sustained release of injectable zinc-recombinant hirudin suspensions: development and validation of in vitro release model.

In humans, recombinant hirudin (rHir), an anticoagulant protein, has a relatively short half-life (about 1 h). Therefore, a rHir formulation with sustained biological activity was previously proposed to result from complexing zinc salts and rHir (Zn-rHir). The purpose of this paper is to introduce and validate an in vitro release model for subcutaneous Zn-rHir formulations. In glass vials the formulations were suspended in agarose gel (2%) and coated with an extra layer of protein-free agarose. The agarose layers were covered with receiver solution, either buffered solutions (HEPES or PBS, pH 7.4) or human serum. To validate the release model and to demonstrate its potential to discriminate between different formulations, several commercial insulin and Zn-insulin formulations were also tested. The release profiles were evaluated by statistical moment analysis (mean times). Only in HEPES buffer was good discrimination between the investigated insulin formulations observed. The mean times of in vitro release of the insulin formulations and the proposed duration of their biological activities were in correlation. Low discrimination was found in PBS. For rHir, clear discrimination between the investigated rHir formulations was achieved in HEPES buffer, whereas low discrimination was found in PBS or in serum. The developed release model may be a sensitive in vitro test to assure the quality of subcutaneous insulin and rHir formulations, and may also be applicable to assess other slow-release protein and low molecular weight drug injectables.

Enhanced potency of human calcitonin when fibrillation is avoided.

The peptide hormone calcitonin (CT) is a potent drug for the therapy of different bone diseases. Salmon CT (sCT) is reported to be more active than human CT (hCT). Human CT, but not sCT, has a strong tendency to aggregate and fibrillate in aqueous solutions. Recent investigations of the fibrillation mechanisms contributed to the development of hCT solutions in which fibrillation is inhibited. Taking into consideration these new findings, we tested the relative activities of hCT handled so as to avoid aggregation/fibrillation, sCT handled in exactly the same way, and hCT handled carefully but without regard to possible fibrillation (denoted R-hCT). The effect of the CTs on bone resorption by isolated osteoclasts was measured. This assay measures the activity of interest (bone resorption) by the cell (the osteoclast) at which therapy is directed. The concentration that inhibits 50% of resorption (EC50) for hCT is 10(-5)-10(-4) pg/mL, compared with 10(-2)-1 pg/mL for R-hCT and 10(-3)-10(-1) pg/mL for sCT. The results show that when aggregation and fibrillation are avoided, hCT at the EC50 is 2-4 orders of magnitude more active than R-hCT. Thus, earlier reports of lower potency of hCT compared with sCT may have been based on inadvertent use of partially aggregated/fibrillated samples of hCT. This finding may have implications for the dose and dosage forms advised for human therapy.

Formulation of sustained release aqueous Zn-hirudin suspensions.

Sustained release formulations for recombinant hirudin (rHir), an anticoagulant thrombin-specific inhibitor, were developed. Zn-rHir suspensions were formed by precipitation with zinc salts at neutral pH. Characterization of protein precipitation was by UV analysis, capillary electrophoresis (CE), zinc analysis, light and electron microscopy, and particle size analysis. The precipitation of aqueous rHir solution with ZnCl(2) solution at neutral pH resulted in Zn-rHir suspensions. Optimum yields of pelletized Zn-rHir were obtained between pH 7.0 and 7.4. For complete precipitation ( approximately 100%) a molar ratio of zinc to rHir of >28 was necessary. As shown by electron microscopy, the smallest resolvable unit of Zn-rHir suspensions was 20 nm. Agglomerates of up to 200 microm were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr(2), ZnI(2), Zn(NO(3))(2) and ZnSO(4) instead of ZnCl(2). ZnSO(4) showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl(2). Upon storage the pelletized protein content of the ZnCl(2) based precipitates was stable ( approximately 95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO(4) based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO(4) based precipitates to hexagonal basic zinc sulfate plates and free rHir. The driving force is the lower aqueous solubility of basic zinc sulfate as compared to the higher solubility of basic zinc chloride.

Ultraviolet Resonance Raman spectroscopy used to study formulations of salmon calcitonin, a starch-peptide conjugate and TGF-ß3.

Ultraviolet Resonance Raman (UVRR) spectroscopy with excitation at 244 nm was investigated here as a possible useful tool for fast characterization of biopharmaceuticals. Studies were performed on three protein drugs: salmon calcitonin (sCT), starch-peptide conjugate, and transforming growth factor-ß3 (TGF-ß3) adsorbed onto solid granules of tricalcium phosphate (TCP). Secondary structure of sCT was investigated for solutions of 0.5mg/mL up to 200mg/mL, regardless of the turbidity or aggregation states. An increase in ß-sheet content was detected when sCT solutions aggregated. UVRR spectroscopy also detected a small amount of residual organic solvent in a starch-peptide conjugate solution containing only 40 µg/mL of peptide. UVRR spectroscopy was then used to characterize a protein, TGF-ß3, adsorbed onto solid granules of TCP at 50 and 250 µg/cm(3). This study shows that UVRR is suitable to characterize the protein formulations in a broad range of concentrations, in liquid, aggregated, and solid states.

Stability of seasonal influenza vaccines investigated by spectroscopy and microscopy methods.

The stability of different seasonal influenza vaccines was investigated by spectroscopy and microscopy methods before and after the following stress-conditions: (i) 2 and 4 weeks storage at 25°C, (ii) 1 day storage at 37°C and (iii) one freeze-thaw cycle. The subunit vaccine Influvac (Solvay Pharma) and the split vaccine Mutagrip (Sanofi Pasteur) were affected by all stresses. The split vaccine Fluarix (GlaxoSmithKline) was affected only by storage at 25°C. The virosomal vaccine Inflexal V (Berna Biotech) was stable after the temperature stresses but aggregated after one freeze-thaw cycle. This study provides new insights into commercial vaccines of low antigen concentration and highlights the importance of using multiple techniques to assess vaccine stability.

Comparative study of human and salmon calcitonin secondary structure in solutions with low dielectric constants.

Molecular conformations of salmon (sCT) and human (hCT) calcitonin in media with different concentrations of methanol/water and trifluoroethanol/water have been investigated by fluorescence, circular dichroism (CD) and infrared spectroscopy techniques. In these media, sCT and hCT adopt an alpha-helical structure comprising up to 40-48% of the amino acids. CD experiments reveal that for both peptides, the ordering of the Cys1-Cys7 disulfide link and the alpha-helix formation can be distinguished. Disulfide bond ordering is similar in both calcitonins. sCT adopts the alpha-helical structure more readily than hCT, as solvent polarity is reduced. In 2,2,2-trifluoroethanol (TFE)/water mixtures the alpha-helix formation for both hCT and sCT is a two-step process. The first alpha-helix formation step occurs at lower TFE concentrations (less than 11 mol% TFE for hCT and 6 mol% TFE for sCT). The second alpha-helix formation step represents 50 and 23% of the whole conformational change for hCT and sCT, respectively. Tyrosine fluorescence measurements correlate with the far ultraviolet CD changes associated with the peptide backbone. hCT is seen to adopt a left-handed, extended conformation in aqueous media below -50 degrees C.

Resonance energy-transfer and fluorescence intensity studies of the transport of liposome-encapsulated molecules into isolated mouse liver nuclei.

We present evidence that liposomes (composed of egg yolk L-alpha-phosphatidylcholine/phosphatidylethanolamine/cholesterol, in a molar ratio of 4:5:1) fuse with isolated mouse liver nuclei at low pH. Using the resonance energy-transfer assay, we determined the rate and extent of liposome and nuclear membrane lipid mixing. Fusion was substantial when the pH was below 5. The half-time of lipid mixing decreased by acidification of the solvent, reaching about 2 min at pH 4.5. In order to study the transport of the liposome-aqueous contents to the interior of the nuclei during the process, we developed fluorescence assays in which fluorescein isothiocyanate labeled dextrans of 150 kDa molecular mass (FITC-D150) were encapsulated in liposomes. These liposomes also included in their bilayers the fluorescent lipid N-tetramethylrhodamine-L-alpha-dipalmitoylphosphatidylethanolamine (N-Rh-DPPE). After incubation of these liposomes with mouse liver nuclei (pH 4.5, 37 degrees C, 30 min), we measured the fluorescence spectra of a suspension of washed nuclei and of nuclei treated by the detergent Triton X-100 (membrane-denuded nuclei). These Triton X-100 treated nuclei had no N-Rh-DPPE fluorescence while they showed a FITC-D150 fluorescence which amounted to 20% of that of the intact nuclei. In another assay, a laser beam was focused on single nuclei by a microscope epiexcitation device. The variation of the N-Rh-DPPE and FITC-D150 fluorescence with the nuclear radius was determined with the microphotometric attachment of the microscope.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysozyme-induced fusion of liposomes with erythrocyte ghosts at acidic pH.

Lysozyme that was covalently bound to the outer surface of sonicated vesicles induced fusion of the vesicles with human white erythrocyte ghosts. The kinetics of membrane mixing were evaluated by the resonance-energy-transfer method using L-alpha-dipalmitoyl phosphatidylethanolamine labeled at the free amino group with the energy donor 7-nitro-2,1,3-benzoxadiazol-4-yl or with the energy acceptor tetramethylrhodamine. The equilibrium state after fusion was characterized by using fluorescence photobleaching and recovery techniques. Rates and equilibrium percentages of fusion were maximal at the pH optimum of the enzyme, and rates were strongly reduced by the addition of N,N',N''-triacetylchitotriose, a competitive inhibitor of lysozyme. An apparent activation energy of 28 +/- kcal/mol was obtained for the lipid-mixing process. At 37 degrees C, the fusion half-time was 0.5 min. After 30 min at 37 degrees C, 40% of the labeled lipids initially present in the fusion mixture had a lateral diffusion constant, D, of 1.1 +/- 0.5 X 10(-9) cm2 X sec-1 in the ghost membrane. The strong induction of fusion at the lysozyme pH optimum was not observed in the absence of lysozyme or when free lysozyme was added to the solution. Bound lysozyme did not induce fusion of electrically neutral liposomes with each other. These observations indicate that it is the liposome-bound lysozyme that induces fusion between liposomes and erythrocyte ghosts.

Porous tricalcium phosphate and transforming growth factor used for anterior spine surgery.

Harvesting autologous bone graft from the iliac crest is associated with considerable secondary morbidity. Bone graft substitutes such as porous ceramics are increasingly used for spinal surgery. This paper presents the results of an animal study in which beta-tricalcium phosphate (beta-TCP) bone substitutes were used for anterior spinal surgery in sheep and baboons. The presented baboon study also investigated the effect of impregnating the ceramic material with transforming growth factor (TGF). In the first study, using the sheep model, a stand-alone instrumented anterior fusion was performed. The animals were randomized into three treatment groups: autologous bone, beta-TCP granules, and sham group. The results were analyzed biomechanically and histologically at three survival intervals: 8, 16 and 32 weeks. An additional animal group was added later, with ceramic pre-filled implants. In the second study, a baboon model was used to assess the osteointegration of a 15-mm-diameter porous beta-TCP block into the vertebral body. The experiment was partially motivated by a new surgical procedure proposed for local bone graft harvest. Three treatment groups were used: beta-TCP plug, beta-TCP plug impregnated with TGF-beta3, and a sham group with empty defect. The evaluation for all animals included computer tomograms at 3 and 6 months, as well as histology at 6 months. In the sheep model, the mechanical evaluation failed to demonstrate differences between treatment groups. This was because massive anterior bone bridges formed in almost all the animals, masking the effects of individual treatments. Histologically, beta-TCP was shown to be a good osteoconductor. While multiple signs of implant micromotion were documented, pre-filling the cages markedly improved the histological fusion outcomes. In the baboon study, the beta-TCP plugs were completely osteointegrated at 6 months. For the group that used ceramic plugs impregnated with TGF-beta3, no incremental advantage was seen as a result of this particular application. However, TGF-beta3 is a potent growth factor at a very low dose. Not only does it speed up the ceramic material resorption, but it is also responsible for massive regional new bone formation. More experiments are required to better understand the biological effects of this growth factor in relation to bone formation, and to be able to take clinical advantage of them.

The structure and mechanism of formation of human calcitonin fibrils.

Turbidity measurements of the kinetics of human calcitonin (hCT) fibrillation showed a linear dependence of the logarithm of fibrillation time (the time the sample is not fibrillated) and the logarithm of hCT concentration. This ln/ln plot linearity and electron microscope observations of fibrils indicate that the fibrillation process can be explained by the double nucleation mechanism that was proposed for the gelation of sickle cell hemoglobin (Ferrone, F. A., Hofrichter, J., Sunshine, H. R., and Eaton, W. A. (1980) Biophys. J. 32, 361-380). Circular dichroism, fluorescence, and infrared spectroscopy studies of fibrils showed that hCT molecules have alpha-helical and beta-sheet secondary structure components. A model for the structure of hCT molecules in fibrils is proposed.

Conformation and self-association of human recombinant transforming growth factor-beta3 in aqueous solutions.

The transforming growth factors-beta (TGF-beta) are important regulatory peptides for cell growth and differentiation with therapeutic potential for wound healing. Among the several TGF-beta isoforms TGF-beta3 has a particularly low solubility at physiological pH and easily forms aggregates. A spectroscopic structural analysis of TGF-beta3 in solution has thus been difficult. In this study, circular dichroism spectroscopy was used to determine the secondary structural elements of TGF-beta3. In addition, the aggregation of TGF-beta3 was investigated systematically as a function of pH and salt concentration using a rapid screening method. Sedimentation equilibrium and sedimentation velocity analysis revealed that TGF-beta3 exists predominantly in two major forms: (i) monomers in solution at low pH and (ii) large precipitating aggregates at physiological pH. Under acidic conditions (pH < 3.8) the protein was not aggregated. At pH approximately 3.9, a monomer right arrow over left arrow dimer equilibrium could be detected that transformed into larger aggregates at pH > 4.1. Aggregation was pronounced in the pH range of 4.3 < pH < 9.8 with the aggregation maximum between pH 6.5 and 8. 5. The aggregation process was accompanied by a structural change of the protein. The CD spectra were characterized by an isodichroic point at 209.5 nm indicating a two-state equilibrium between TGF-beta3 dissolved in solution and aggregated TGF-beta3. Aggregated TGF-beta3 showed a higher beta-sheet content and lower beta-turn and random coil contributions compared with monomeric TGF-beta3. Both the solution structure and the aggregate structure of TGF-beta3 were different from the crystal structure. This was in contrast to TGF-beta2, which showed very similar crystal and solution structures. Under alkaline conditions (pH > 9.8) the turbidity disappeared and a further conformational change was induced. The pH dependence of the TGF-beta3 conformation in solution in the range of 2.3 < pH < 11. 0 was reversible. Aggregation of TGF-beta3 was, furthermore, influenced by the presence of salt. For pH > 3.8 the addition of salt greatly enhanced the tendency to aggregate, even in the very basic domain. Under physiological conditions (pH 7.4, cNaCl = 164 mM) TGF-beta3 has almost the highest tendency to aggregate and will remain in solution only at nanomolar concentrations.