WATER-SOLUBLE ELASTIN AS A COSMETIC MATERIAL 15 RESULTS AMINO ACID COMPOSITION The amino acid composition of HAPA-elastin was determined by partial hydrolysis of pig aorta and purifi cation of elastin peptide mixtures, as previously reported (15,16). The data are summarized in Table I. For all three elastin samples studied, the total content of Gly, Pro, Ala, and Val was in the range of 80.1–81.5%. Conversely, the contents of acidic amino acid residues, Asp and Glu, and basic amino acid residues, Lys, His, and Arg, were rela- tively lower and were in the ranges of 2.5–3.6% and 1.3–1.7%, respectively. It is well known that elastin contains two characteristic amino acids, Des and Ide. Together, Des and Ide residues formed 0.2–0.4% of the total amino acids in the HAPA-elastin. Our results are in good agreement with the amino acid composition of pig elastin obtained through gene sequencing, as reported in the National Center for Biotechnology Information data- base (NCBI, GenBank: BAP76077.1). Three main amino acid components of HAPA- elastin (Gly, Ala, and Pro) are also present in high amounts in the human epidermis. It has therefore been suggested that this water-soluble elastin is a useful cosmetic material, which can supply the epidermis with the abovementioned benefi cial amino acids (5–7,17). COACERVATION MEASUREMENTS HAPA-elastin showed an apparent coacervation property at a protein concentration of 100 mg/ml (Figure 2). The HAPA-elastin solution was initially clear but became turbid Table I Amino acid composition of HAPA-elastin and elastin peptides from porcine aorta. Amino acid Residues / 1,000 total residues HAPA-elastin Elastin peptides from porcine (15) Elastin peptides from porcine (16) Hyp 9 6 11 Asp 9 3 6 Thr 4 21 14 Ser 6 11 11 Glu 27 23 19 Pro 114 107 117 Gly 336 325 330 Ala 234 242 234 Val 131 128 120 Met 1 0 0 Ile 16 16 18 Leu 53 53 54 Tyr 11 21 16 Phe 26 29 33 Des + Ide* 4 2 3 His 7 0 1 Lys 8 8 6 Arg 2 7 6 *Des, desmosine Ide, isodesmosine.

JOURNAL OF COSMETIC SCIENCE 16 at 37°C, as the temperature of the solution was increased from 5°C to 65°C. Maximum turbidity was observed at 45°C. When the temperature of the solution was decreased from 65°C to 5°C, turbidity decreased in a temperature-dependent manner, and the solu- tion became clear again at 5°C. Consequently, the profi les for change in turbidity with temperature were almost identical for both the heating and cooling cycles. This indicates that the coacervation of HAPA-elastin may be reversible however, the reversibility is temperature dependent. As shown in Figure 2, HAPA-elastin did not show a complete coacervation property at 50 mg/ml, indicating that the coacervation of HAPA-elastin is concentration dependent. SEPARATION BY COACERVATION HAPA-elastin is prepared from pig aorta and contains a mixture of elastin degradation products. To understand in detail the characteristics of each component of the elastin degradation mixture, HAPA-elastin was further separated into its individual components by using a phase separation method and gel fi ltration chromatography. The solution was separated into two layers via coacervate formation. Starting with 24 g of HAPA-elastin, we obtained 20 g of fraction 1 (supernatant) and 2.4 g of fraction 2 (precipitate), which translates to 89.3% and 10.7%, respectively, of each fraction. The molecular weight of each fraction was determined by gel fi ltration chromatography (Figure 3). The molecular weights of the fractions ranged from 1,350 to 670,000 Da, with those of fractions 1 and 2 ranging from 1,000 to 44,000 Da and 2,000 to 670,000 Figure 2. Coacervation property of HAPA-elastin. The concentration of HAPA-elastin was either 50 or 100 mg/ml.