Hydroxyproline colorimetric procedures

A new approach to hydroxyproline assay is a recently introduced enzymatic procedure based on the ability of a multiple-enzyme system in adapted Pseudomonas strains of bacteria to degrade hydroxyproline (R7). Also, a standard colorimetric procedure has been adapted for use in an AutoAnalyzer apparatus (G5). Automatic amino acid analyzers can be easily programmed to isolate hydroxyproline from its more usual place in the shoulder of the aspartic acid peak (see M15). 

Fio. 3. Absorption spectra of chromophores from 125 /jg of authentic hydroxyproline and protein hydrolysate of 1 ml of plasma. Chromophores were concentrated by adding 0.6 ml of water to the final ethanol-sulfuric acid-toluene-Ehrlich s reagent mixture of the colorimetric procedure, shaking the mixture, and separating the water-ethanol-acid phase the latter was then diluted to 3.0 ml with ethanol (L9). 

Cardiac muscle hydroxyproline, measured by a simple colorimetric procedure with inadequate specificity in this situation, increased slightly beyond age 35-40, while a similar rise was seen in the hydroxyproline of aortic tissue (C9). Other studies using identical methods showed no change with age in aortic and pulmonary artery hydroxyproline (Bll, K2). The influence of both age and cardiac hypertrophy on cardiac... 

It is important to be able to determine which chromatography column fractions contain polysaccharides and, specifically, which fractions contain hexosyl, pentosyl, or uronosyl residues. It is also important to detect in the fractions the presence of proteins and the presence of the specific amino acid characteristic of wall proteins, hydroxyproline. The detection of these substances is carried out by facile and sensitive colorimetric procedures. Although these reactions are not discussed here, the most frequently used colorimetric assays in our laboratory are the anthrone assay for detection of hexosyl residues 50), the orcinol assay for detecting pentosyl residues 50), the m-hydroxy-diphenyl assay for detection of uronosyl residues 35), the Lowry assay for detection of proteins 90), and the Kivirikko and Liesmaa assay for the detection of hydroxyprolyl residues 76). 

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