Hydroxyproline isolation

As indicated in Figure 2, when minces of tumor obtained from normal and ascorbic acid-deficient animals were incubated with C14-pro-line, much more radioactivity was incorporated into the collagen of the normal tissue. When the specific radioactivities of the isolated imino acids were examined (Table HI), several conclusions were possible. In the experiments with granuloma from normal animals and C14-proline, the values for hydroxyproline were not far from those of proline. With the scorbutic granulomas, the specific activity of the isolated proline was not greatly reduced, compared to that obtained from the normal granuloma. In contrast, the specific activity of the hydroxyproline isolated from the deficient tissues was markedly reduced. Similar results were obtained in the studies with tritiated proline. Thus, the specific activity of the proline isolated from the deficient granuloma was only moderately reduced, whereas the specific activity of the hydroxyproline was extremely low. This observation may be explained in terms of a dual-pathway mechanism of proline incorporation, considered below. 

The striking fall in the specific activity of the collagen hydroxyproline isolated from deficient granulomas observed in the present study (Table HI) suggests an alternative scheme of collagen synthesis. 

Table IV. Ratios of Tritium to Carbon-14 for Proline and Hydroxyproline Isolated fron Collagen0...

FIGURE 4.4 The structures of several atniuo acids that are less cotntnou but nevertheless found in certain proteins. Hydroxylysine and hydroxyproline are found in connective-tissue proteins, pyroglutatnic acid is found in bacteriorhodopsin (a protein in Halohacterium halohium), and atninoadipic acid is found in proteins isolated from corn. 

Other workers began to study the structure of gramicidin. Christensen and coworkers12 isolated crystalline tryptophane and leucine from a hydrolysate. They found no evidence for a fatty acid component and established that phenylalanine, proline and hydroxyproline were absent from a hydrolysate. These workers isolated alanine diox-pyridate from a hydrolysate and also established that gramicidin contained a compound with vicinal hydroxy and amino groups. They speculated that this compound might be serine or isoserine and proposed that gramicidin contains two tryptophane, 2 leucine, 2 or 3 alanine and 1 hydroxyamino residues or a multiple of this composition. 

Plaquet et al. (PI) found in the urine of rachitic children peptides consisting of proline, hydroxyproline, and glycine, which they believed to be the products of collagen degradation. Two similar peptides containing considerable amounts of proline and hydroxyproline were isolated from the urine of a patient with rheumatoid arthritis by Mechanic et al. (Ml). One of these peptides consisted of three proline, two hydroxyproline, and nine glutamic acid residues, the second one consisted of four proline, four hydroxyproline, and one glutamic acid residues. The N-terminal amino acid in the first peptide was demonstrated to be hydroxyproline. 

By means of a procedure described above, Hanson and Fittkau (HI) isolated seventeen different peptides from normal urine. One of them, not belonging to the main peptide fraction, consisted of glutamic acid, and phenylalanine with alanine as the third not definitely established component. The remaining peptides contained five to ten different amino acid residues and some unidentified ninhydrin-positive constituents. Four amino acids, i.e., glutamic acid, aspartic acid, glycine, and alanine, were found in the majority of the peptides analyzed. Twelve peptides contained lysine and eight valine. Less frequently encountered were serine, threonine, tyrosine, leucine, phenylalanine, proline, hydroxyproline, and a-aminobutyric acid (found only in two cases). The amino acid composi-... 

MI. Mechanic, G., Skupp, S. J., Sailer, L. B., and Kibrick, A. C., Isolation of two peptides containing hydroxyproline from urine of a patient with rheumatoid arthritis. Arch. Biochem. Biophys. 86, 71-76 (1960). 

Isolation of individual amino acids started about 1820 by 1904 all of the naturally occurring amino acids in proteins had been isolated except methionine (Mueller, 1922) and threonine (Rose, 1937). One of the earliest methods for the separation of amino acids was through the differential volatility of their methyl or ethyl esters (Emil Fischer, 1901). This approach led to the discovery of valine, proline, and hydroxyproline. [In the 1970s Fischer s method was modified for microanalysis of proteins, separating the amino acid esters by gas phase chromatography. Separation is now usually performed by hplc (high pressure liquid chromatography).]... 

S,4/ )-4-Hydroxyproline also served as starting material for the preparation of crotanecine, isolated from alkaloids of Crotalaria sp. Compound 62 was obtained in fifteen steps in 4% overall yield (84H2735). 

The red pigment isolated in crystalline form from the reaction products of 1-methyl-3,4-benzoquinone and 4-hydroxyproline ethyl ester was identified as 4-(4 -hydroxy-2 -carbethoxy-l -pyrrolidyl)-5-methyl-l,2-benzoquinone (115).283 This highly colored compound should probably be formulated as a zwitterion (116). Similar pigments can be obtained by the interaction of o-benzoquinone with proline, hydroxyproline, pyrrolidine, and glycine. Suzuki has shown... 

S.N. Bhattacharyya, M.A. Passero, R.P. DiAugustine and W.S. Lynn, Isolation and characterization of two hydroxyproline-containing glycoproteins from normal animal lung lavage and lamellar bodies, J. Clin. Invest. 55 (1975) 914-920. 

In 1899 Fischer turned his attention to the study of proteins, wishing to understand their chemical structures. It was known at that time that proteins were composed of amino acids, and thirteen naturally occurring ones were identified. Fischer was able to isolate via the hydrolysis of proteins three additional naturally occurring amino acids valine, proline, and hydroxyproline. Amino acids exhibit stereoisomerism, and Fischer was able to separate individual forms from mixtures of stereoisomers for several of these compounds. 

McNamara, M. K., and Stone, B. A. (1983). Isolation, characterization and chemical synthesis of a galactosyl-hydroxyproline linkage compound from wheat endosperm arabinogalactan-peptide. Lebensm. Wiss. Technol. 14 182-187. 

Table I. Mole Ratio of Proline to Hydroxyproline in Collagen Isolated from Granuloma of Normal and Scorbutic Guinea Pigs0...

In an attempt to learn whether collagen hydroxyproline synthesized in granuloma minces incubated with tritiated proline contained tritium at the 4 position, we converted such tritiated hydroxyproline to the corresponding N-carbobenzoxy derivative and then oxidized this derivative with chromic acid to N-carbobenzoxy-4-ketoproline according to the procedure of Patchett and Witkop(29). In most of these experiments very little tritiated water was formed although some tritiated water was detected in several experiments, the small amount formed could logically be attributed to adventitious oxidative reactions or perhaps to enolization of ketoproline. The evidence was therefore consistent with the absence of tritium at carbon atom 4 of the isolated hydroxyproline. 

Ebert and Prockop (8) carried out similar studies with 3,4-triti-ated proline in chick embryos, and reported ratios of 0.73 and 0.74. Information supplied by Prockop (32) indicates that this material was from a batch (New England Nuclear Corp.) different from those used by us. They converted the isolated hydroxyproline to pyrrole and found ratios of the H3/C14 values for pyrrole to hydroxyproline of 0.61 and 0.64 (these values were corrected for loss of carboxyl C14, assuming random labeling). We have recently converted the H3-hydroxyproline obtained in our experiments with the new batch of 3,4-tritiated proline to pyrrole (32), and obtained values of 0.70 and 0.73. However, conversion of this hydroxyproline to N-carbobenzoxy-4-ketoproline did not lead to a significant decrease in specific activity, suggesting that little tritium was present at carbon atom 4 of the isolated hydroxyproline. 

If this scheme is correct, it should be possible to obtain conditions under which protein synthesis but not hydroxylation is blocked. That this may be possible experimentally is suggested by the results of recent experiments (24) in our laboratory, which have shown that in the presence of puromycin, the formation of collagen hydroxyproline determined in carageenin tumors as described here is markedly reduced, but that hydroxylation (as determined by the formation of tritiated water and tritiated hydroxyproline) continues. Further work—especially efforts directed at isolation of intermediates—are in progress. 

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