Quantitative assay for hydroxylysine in protein hydrolyzates

✍ Scribed by Nelly Blumenkrantz; Darwin J. Prockop

• Publisher: Elsevier Science
• Year: 1971
• Tongue: English
• Weight: 319 KB
• Volume: 39
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(71)90461-1

✦ Synopsis

Collagen and related glycoproteins are the only animal proteins which contain significant amount's of the hydroxyamino acids hydroxyproline and hydroxylysine. A number of chemical procedures for the quantitative assay for hydroxyproline have been developed, and these assay procedures have been used extensively on the studies of biosynthesis and metabolism of collagen (see reference 1 for review). Similar studies on the biosynthesis of hydroxylysine have in part been hampered by the lack of rapid and specific assays for this amino acid.

In the present report we present an assay for hydroxylysine which is based on periodate oxidation. Periodate will oxidize a number of amino acids such as scrine, threonine, proline, and hydroxyproline, and periodate oxidation of both serine and hydroxylysine produces ammonia and formaldehyde (2-4). Part of the specificit#y of the present assay was based on the fact that periodate oxidation of hydroxylysine also produces glutamic semialclehyde and Al-pyrroline-5-carboxylic acid which on further oxidation will form a color with p-dimethylaminobenzaldehyde (Fig. 1). Initial experiments indicated t'hat proline gave rise to a similar chromophorc in the assay and therefore a preliminary separation of samples by thin-layer chromatography was introduced into the procedure.

MATERIALS

Ground-glass t&es with stoppers or culture tubes, screw-capped with Teflon lillers (Kimaz)

, 200 X 25 mm. Tubes were marked with a diamond knife in order to calibrat'e them to a volume of about 8 ml.