2.3. Amino Acid Composition

About 10 mg of freeze-dried powdered sample of host coral tissue or mucus, or a piece of dried glass fiber filter containing symbiotic algae, was treated in 6 N HCl at 110 °C for 12 h to hydrolyze the peptides into free amino acids. The hydrolysate was dried under a stream of N₂ at 40 °C and then redissolved in an appropriate amount of ultrapure water (Milli-Q, Merck-Millipore, Burlington, MA, USA. This solution was injected into a high-performance liquid chromatography (HPLC) system (NANOSPACE SI-1, Shiseido, Tokyo, Japan) equipped with an autosampler (SIL-10, Shimadzu Scientific Instruments Inc., Kyoto, Japan), a semi-micro reverse-phase separation column (CAPCELL PAK C18, Shiseido, Tokyo, Japan), and a fluorometric detector, in which pre-column derivatization of amino acids with o-phthaldialdehyde [68] was performed automatically after injection. The following amino acids were quantified: alanine (Ala), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), aspartic acid + asparagine (Asx), threonine (Thr), β-alanine (β-Ala), serine (Ser), methionine (Met), glutamic acid + glutamine (Glx), phenylalanine (Phe), histidine (His), tyrosine (Tyr), arginine (Arg), ornithine, and lysine (Lys). Cysteine (Cys), proline (Pro), and tryptophan (Trp) could not be quantified using this method, and Met may also have been partially lost during the acid hydrolysis. The total amino acid concentration was defined as the sum of all amino acids detected. The amino acid composition is given as the mole percentage of each amino acid in total amino acids.