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SynopsisThe temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (~-Val~-~-Pro~-Gly~-~-Val~-Gly~)~ in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data a t lower water composition, allow construction of a phase-structure diagram of the polypentapeptide-water system. The molecular-weight dependence of phase change (coacervation) is included. The volumecomposition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity. INTRODUCTIONThe polypentapeptide of elastin (~-Val'-~-Pro~-Gly~-~-VaI~-Gly~) where n = 11 in the pig' and 13 in the chick (L. B. Sandberg, private communication) precursor protein of the elastic fiber, has been synthesized with n 'v 200.2 This polymer is soluble in all proportions in water below 20"C, but on raising the temperature aggregation occurs and the aggregates settle to form a dense viscoelastic phase called a coacervate. The process of coacervation is readily reversible and the coacervate is a two-component system that, as will be shown here, is by weight 63% water and 37% polypentapeptide at 30°C. The high molecular weight polypentapeptide, once cross-linked for example by y-irradiation at 15 to 20 Mrad, is elastomeric with an elastic modulus and a thermoelasticity curve that are similar to those of fibrous e l a~t i n .~ Fibrous elastin is the resulting matrix when elastic fibers are stripped of a fine microfibrillar coat of glycoprotein. Because of this similarity of the thermoelasticity curves and because the polypentapeptide is the most striking primary structural feature of the sole precursor protein of fibrous elastin,' studies on the polypentapeptide of elastin provide valuable insight into the mechanism of biological elasticity. In its own right the polypentapeptide of elastin is a promising new elastomeric biomaterial. Reported here are the phase-structure diagram for the polypentapeptide-water system and thermal coefficients of coacervate expansion and composition. The phase-structure diagram provides in a single figure a summary of the phase and polypeptide structure behavior as a function of water content and temperature. The diagram should prove useful in consideration of numerous physical characterizations, and theoretical considerations of the viscoelastic coacervate and the thermal coefficients are of specific interest in analyzing thermoelasticity data on the y-irradiation cross-linked polypentapeptide coacervate. Representative CD data that demonstrate the structural transitions are also reported. These are further discussed with respect to nmr and dielectric relaxation studies. and Boc-VP-OBzl was obtained using the water-soluble carbodiimide method.8 After hydrogenating I1 and acid deblocking 111, Boc-GVG-OH was coupled with H-V...

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of VaI-Gly-VaI-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at ~10 -8 M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucylphenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.Chemotactic activity has been associated with several components of the extracellular matrix including collagen (18), fibronectin (1, 5, 17), laminin (27), peptides of insoluble elastin (6, 22,23), and tropoelastin (23). The sites of chemotactic activity on collagen (16) and fibronectin (1, 25) have been partially localized, however the chemotactically active regions of laminin and elastin have not yet been established. The present study was undertaken to identify the site(s) of chemotactic activity on elastin.In approaching the molecular basis of the chemotactic activity of elastin peptides two points seemed important: first, the presence of fibroblast chemotactic activity in tropoelastin (23), the soluble precursor of insoluble elastin (20), suggested that the lysine-derived cross-links characteristic of insoluble elastin are not essential for the chemotactic activity of elastin peptides; and second, the presence of repeating peptide sequences in tropoelastin (4, 19) directed us to look at these peptide repeats as the possible source of the chemotactic activity. In this report, we present evidence that some of the chemotactic activity of elastin is associated with Val-Gly-ValAla-Pro-Gly (VGVAPG),~ a hexamer that repeats six times in one tryptic fragment of porcine tropoelastin (20). MATERIALS AND METHODS Preparation of Elastin-derived Peptides:Bovine ligament elastin, obtained from Elastin Products (St. Louis, MO), was solubilized with porcine pancreatic elastase (Sigma Chemical Co., St. Louis, MO) by incubation a...

Differential scanning calorimetry studies of the effect of NaCl on protein-based polymer self-assembly has been carried out on six elastin-based synthetic sequential polypeptides--i.e., the polypentapeptide (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n and (L-Val1-L-Pro2-L-Ala3-L-Val4-Gly5)n; the polytetrapeptide (L-Val1-L-Pro2-Gly3-Gly4)n and its more hydrophobic analogue (L-Ile1-L-Pro2-Gly3-Gly4)n; and the polynonapeptide (a pentatetra hybrid), (L-Val1-L-Pro2-Gly3-L-Val4-Gly5-L-Val6-L-Pro7-Gly8-Gly9++ +)n. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order of the polypentapeptide, as the temperature is raised from below to above that of the transition, has been repeatedly observed using different physical characterizations. In the present experiments, it is observed that the transition temperatures of the polypeptides studied are linearly dependent on NaCl concentration. The molar effectiveness of NaCl in shifting the transition temperature delta Tm/[N], is about 14 degrees C/[N], with the dependence on peptide hydrophobicity being fairly small. Interestingly, however, the delta delta Q/[N] does depend on the hydrophobicity of a polypeptide.

Six syntheses of gramicidin A have been carried out, each with 90% lC enrichment of a single carbonyl carbon these being the formyl, Val-1I Trp-9, and