Specific Determination of Hydroxy-L-Proline in Biological Materials.

Abstract

Paper chromatographic studies on the amino acid metabolism of animal tissues in vitro (1) have shown that marked changes in amino acid content of synthetic medium JI 1 50 ( 2.3 ) occur during the cultivation period.Medium M 150 contains 60 ingredients, including 20 amino acids, in a modified Tyrode's solution(4) and its complexity made it difficult to detect changes in amino acids that were present in low concentrations or that did not separate completely on the chromatograms. Particular difficulty was encountered with hydroxyproline, which reacted only slightly on development with ninhydrin( S ) , isatin ( 6 ) , vanillin (7) or isatin and p-dimethylaminobenzaldehyde (8). For this reason, an effort was made to develop a selective method for hydroxyproline that could be applied in the presence of high concentrations of other amino acids. The present communication reports such a specific method, based on a modification of the conventional ninhydrin procedure. The application of this method to complex synthetic media and to deproteinized chick embryo extract is presented.Samples of media for analysis (5.0 ml) were concentrated to dryness in vacuo over H2S04, reconstituted in 0.2 ml of deionized water and 10 ii portions used without desalting. One-dimensional descending chromatograms (Schleicher and Schuell S o . 597 or Whatman No. 1 paper) were developed for 2 successive 18-hour periods in either n-butanol-acetic acid-water or n-butanol-ethanol-water.The chromatograms were dried at 110°C for 2 to 3 minutes and sprayed with 0.4% ninhydrin in water-saturated n-butanol or 95% ethanol. Full details of the chromatographic procedures have been reported previously ( 1,9,10). Chick embryo extract was prepared by grinding 11-*The cooperation of Mr. C. E. Kerr, of this Department, in making the photographic preparations, is gratefully acknowledged. Methods.~-_-day-old embryos and centrifuging off the tissue pulp( 11). The supernatant extract was adjusted to pH 4.0 with 1 r\' H2S04, precipitated proteins removed by centrifuging, and the pH readjusted to 7.2. Two volumes of acetone were added, the mixture allowed to stand overnight in the refrigerator, and centrifuged. The final supernatant was concentrated to dryness in the usual manner and used for analysis. Synthetic medium M 150 was prepared as described previously( 2,3). All chemicals and reagents were of the highest purity obtainable. Spectrophotometric measurements were made by cutting appropriate areas from the developed chromatograms and fixing them to the inner walls of 1 cm Corex cells. Determinations were then made in a Beckman, Model DC. Ultraviolet examinations were carried out with a laboratory hand lamp (short wave model, 2537Results. Development of a specific color with hydroxy-L-proline and ninhydrin, Previous studies on the determination of phenylalanine in synthetic medium 31 lSO(10) showed that dipping ninhydrin-developed chromatograms in 1 % sodium bicarbonate produced a stable blue color characteristic of this amino acid. At the same time, this alkali tre...

Development of Collagen in the Frog Embryo

“…Apparently, none of the amino acids in gelatin is responsible; gelatin hydrolyzates included routinely in all tests gave values for hydroxyproline close to those generally accepted. Similarly, amino acids which are either absent from or in very low concentration in collagen but which would be expected in hydrolyzates of other proteins-cystine, histidine, phenylalanine, and tyrosine-did not interfere with the hydroxyproline test when they were added either singly or together to gelatin hydrolyzates in amounts up to 25 ,g/ml (however, cf. Martin and Axelrod"7).…”

Biosynthesis of Collagen

“…Reference spots of proline and hydroxyproline were placed at the end of the hydrolysate streak. A descending chromatogram was developed in n-butanol-water-acetic acid (275:100: 75) for 18 hr in the cold room, dried in air at room temperature, and redeveloped for a second 18 hr period (29,30). The imino acids were located by spraying the reference strip with ninhydrin-isatin and developing in an oven at 80-100~C for 10 min.…”