Rapid robust separation of hydroxyproline and proline.

The presence of hydroxyproline, and the determination of the ratio of the secondary amino acids proline to hydroxyproline, in amino acid hydrolysates specifically identifies collagen and collagen peptides. o-Phthalaldehyde, and then 9-fluorenylmethyl chloroformate, were used to carry out sequential prederivatization of amino acid hydrolysates in an in-line high-performance liquid chromatography sample loop. After derivatization, reversed-phase high-performance liquid chromatography with a C-18 ODS Hypersil cartridge column was used to resolve the hydroxyproline and proline from all primary amino acids, with resolution and detection of hydroxyproline and proline within 2.0 and 2.8 min, respectively, at concentrations in the range of picomoles per microliter of derivatized amino acid. The assay has a turnaround time of 10.75 min.     

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.     

Imino acid transport in human diploid fibroblasts.

These studies describe the transport of proline and hydroxyproline in human diploid fibroblasts. Inhibition and kinetic analysis demonstrate that proline is actively transported by the “A” neutral amino acid carrier. Proline transport is Na⁺ dependent and is particularly sensitive to sulfhydryl inhibitors and ouabain. Hydroxyproline is also actively transported but its transport is mediated by a system different from those described previously for other neutral amino acids. Hydroxyproline transport requires the presence of Na⁺ and is sensitive to sulfhydryl inhibitors and ouabain. There is little inhibition of hydroxyproline transport in the presence of other amino acids with the exception of methionine. The methionine inhibition of hydroxyproline transport is of the non-competitive type. Little cross-reactivity was exhibited by the systems which transport proline and hydroxyproline. These studies indicate that human skin fibroblasts do not possess an iminoglycine transport system as has been described for many other tissues. The iminoglycine transport system has been identified as the genetic transport defect in iminoglycinuria. Consequently, skin fibroblasts are not an appropriate system for use in diagnosis of this disorder.     

A new rapid method for quantitating radioactive proline, 4-hydroxyproline,and 3-hydroxyproline

A simple and rapid method for separating proline, 4-hydroxyproline and 3-hydroxyproline was developed by the use of high-voltage electrophoresis. An excellent counting efficiency of proline was achieved by a simple extraction of the labeled material from paper; 98–100% recovery of count rates was obtained, equivalent to recoveries from column chromatography. This method can tolerate high concentrations of salt, acid, and protein in the sample. No carrier is required and multiple samples (up to 15) can be separated on a single sheet and analyzed within 2 h. Serial dilution experiments showed excellent linearity. An average recovery rate of 92% was obtained for samples over a wide range of radioactivity and high sensitivity of the method was demonstrated. This analysis is applicable to protein hydrolysates and to determination of the free amino acids in the presence of protein. Thus, proline, 4-hydroxyproline and 3-hydroxyproline can be quantitated simultaneously in any biological sample.     

Separation and evaluation of the cis and trans isomers of hydroxyprolines: effect of hydrolysis on the epimerization

A procedure has been developed which can detect the hydroxyproline isomers trans-4-hydroxyproline (Hyp), trans-3-hydroxyproline, cis-4-hydroxyproline, and cis-3-hydroxyproline present in hydrolysates of collagens. The method involves hydrolyzing collagen, and reacting the primary amino acids with o-phthaladehyde (OPA) and the hydroxyprolines and proline with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) which combines specifically with secondary amino acids. The proline and hydroxyprolines are then separated by thin-layer chromatography and quantified by using a scanning spectrofluorometer. The method was used to show that both trans-4-L-hydroxyproline and trans-3-L-hydroxyproline were epimerized as a function of hydrolysis time to the cis isomers. An appreciable amount of trans-3-Hyp was degraded. Hydrolysis with 6 N HCl in the presence of 6% trichloroacetic acid gave greater epimerization than the 6 N HCl alone. Alkaline hydrolysis in 0.2 M Ba(OH)2 caused more epimerization of trans-4-Hyp and trans-3-Hyp compared with acid hydrolysis but less degradation, so that alkaline hydrolysis is proposed for the evaluation of trans-3-Hyp, provided that the total of the cis and trans isomers be considered in this case.     

Single-column amino acid analysis of connective tissue proteins and related materials.

A single-column procedure is described which, in general, is useful for the amino acid analysis of any simple or complex protein, but in particular the procedure is useful for the separation and quantitation of the amino acids and amino sugars, if present, in collagen, elastin and related materials from a variety of connective tissues. In addition to the amino acids commonly found in proteins, the system resolves hydroxyproline, hydroxylysine, desmosine, isodesmosine, glucosamine, galactosamine and also a number of other ninhydrin-positive compounds ordinarily not found in acid hydrolysates of proteins. Chromatograms of the analysis of a synthetic mixture of amino acid standards and also collagen and elastin hydrolysates indicate the multiple use of the procedure and the nearbaseline separation of all the amino acids. The basic amino acids are well spread, thus providing flexibility for the isolation and identification of additional ninhydrin-positive components. The analysis, which requires approximately 2 hr and four sodium citrate buffers was performed with a Durrum (D-500) amino acid analyzer.     

Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide

Amino acids are quantitatively determined by precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and reversed-phase high-performance liquid chromatography with photometric detection at 340 nm. Excellent chromatographic resolution of a mixture of the derivatives of 20 amino acids including proline and cystine is achieved within 110 min by linear gradient elution with acetonitrile in 13 mM trifluoroacetate plus 4% (by vol) tetrahydrofuran. The limit of detection is 50 pmol. Amino acid analyses of acid hydrolysates of the several proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multipurpose high-performance liquid chromatographic system.     

Automated fluorometric amino acid analysis: the determination of proline and hydroxyproline.

A fluorometric method for the automated determination of the imino acids proline and hydroxyproline has been developed. The assay is based on the postcolumn reaction of the imino acids with alkaline sodium hypochlorite, which yields oxidation products amenable to detection with fluorogenic amine reagents. The method is simple and can be adapted readily to high-sensitivity amino acid analyzers which use o-phthalaldehyde for detection. As little as 10 pmol proline and 20 pmol hydroxyproline can be determined accurately. Thus the full array of natural imino and amino acids can now be determined on a high-sensitivity amino acid analyzer using o-phthalaldehyde.     

Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.     

Determination of radioactive proline and hydroxyproline by a simple paper-chromatographic procedure

A simple method to separate and determine radioactive proline and hydroxyproline by paper chromatography is described. The localization of the imino acids separation is achieved by direct nondestructing staining with 1-fluoro-2,4-dinitrobenzene dye. The imino acids are quantitated directly by liquid scintillation counting in the presence of paper strips. The method was applied to bone cultures with good reproducibility, sensitivity and linearity over a wide range of radioactivity. The procedure was also tested in fibroblast cultures. The results for hydroxyproline were in good correlation with the widely used method of Juva and Prockop (Juva, K. and Prockop, D.J. (1966) Anal. Biochem. 15, 77–83), in which hydroxyproline is oxidized to pyrrole, and then extracted and purified by column chromatography before counting radioactivity.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20–200 pmol, with equations y = 1.10x − 0.80 (r = 0.999) and y = 1.12x − 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 ± 0.04 and 2.03 ± 0.04 nmol/μg, respectively.     

Purification and Characterization of a Salt-extractable Hydroxyproline-rich Glycoprotein from Aerated Carrot Discs

The salt-extractable hydroxyproline-rich glycoprotein (HRGP) of the cell wall of aerated carrot root discs has been studied by polyacrylamide gel electrophoresis. The predominant proline-labeled protein extractable from the cell wall is rich in hydroxyproline as shown by its specific loss of ³H from proline labeled in position 4 and its shift in electrophoretic mobility after labeling in the presence of an inhibitor of hydroxyproline synthesis. Unlabeled HRGP can be identified by staining gels for carbohydrate. The HRGP has been purified by ion exchange chromatography and CsCl gradient centrifugation. The HRGP consists of about 50% protein and 50% carbohydrate with an overall molecular weight of 86,000. The amino acid composition of the protein portion consists of 50% hydroxyproline, 19% basic amino acids, 12% serine, and 10% tyrosine. This glycoprotein accumulates in a salt-extractable pool in the cell wall beginning between 10 and 20 hours of aeration and may also become incorporated into the nonextractable portion of the cell wall.     

Studies on collagen metabolism in rats. I. Age-related changes in turnover and amino acid composition of the collagen of glomerular basement membrane and tail tendon

To examine the effect of age on the collagen metabolism of glomerular basement membrane (GBM) and tail tendon, both structures were labeled in vivo by the injection of tracer amounts of radioactive proline. The analyses of the labeled proline and, especially, hydroxyproline revealed that synthesis and degradation are reduced in senescent rats. The calculated turnover rate for GBM-collagen is prolonged by about 40% with increased life span. The reduced catabolism is responsible for the age-related thickening of the GBM. In addition, compositional analyses of isolated GBM and tail tendon were performed. Substantial age changes were observed in the amino acid composition of both structures. The hydroxylated amino acids showed a progressive increase with age, while proline and lysine were found to decline.     

Separation of amino acids using ion-paired reversed-phase high-performance liquid chromatography with special reference to collagen hydrolysate

Summary The collagen study includes the analysis of its characteristic amino acids: proline, hydroxyproline, lysine, hydroxylysine. HPLC offers an interesting device if associated with on-line radiometric detection for the determination of radiolabelled amino acids in the case of metabolism studies. To avoid pre or post-column derivatization which may be poorly quantitative in the case of the hydrolysate of unpurified samples, we developed an ion-paired reversed-phase chromatography using a C8 column (econosphere C8 5µm, length: 250 mm, ID: 4.6 mm from Alltech Ass.) and an elution carried out with an acetonitrile gradient in heptane-sulfonate solution. A direct detection at 210 nm was used. Nineteen amino acids were separated within 40 min. Lag time was 7.3 min between hydroxyproline and proline, and 6.9 min between hydroxylysine and lysine. In the case of radiolabelled amino acid, there was a linear correlation (r = 0.92) between HPLC and ion-exchange chromatography.     

Role of oxygen fixation in hydroxyproline biosynthesis by etiolated seedlings

Etiolated maize and soybean seedlings were grown for several days in atmospheres enriched with O¹⁸. Hydroxyproline subsequently isolated from the seedlings by column and thin-layer chromatography was labeled with excess O¹⁸, but proline was not. Control experiments in which seedlings were grown in H₂O¹⁸ and unlabeled atmospheres demonstrated that neither proline nor hydroxyproline was labeled with excess O¹⁸. It was concluded that oxygen fixation is an essential feature of hydroxyproline biosynthesis in these seedlings, and that the hydroxyl oxygen atom in hydroxyproline is derived from molecular oxygen and not from water; similar results have been reported previously for sycamore cell suspensions.     

Single column analysis of amino acids in protein hydrolysates utilizing the fluorescamine reaction

A system is described for the separation of the amino acids commonly found in protein hydrolysates at the picomole level using a single ion exchange column and for their quantitation by the fluorescamine (4-phenylspiro[furan-2 (3H),1′-phthalan]-3,3′-dione) reaction. Three sodium citrate buffers were required for the separation of the amino acids with an analysis time of approximately 3 hr. The amino acids in 1 μg of hydrolyzed bovine serum albumin were separated using a single ion exchange column and were detected in the effluent from the column by the fluorescamine assay. The results were compared with those obtained using a commercial amino acid analyzer and 150 μg of hydrolyzed bovine serum albumin. The chromatogram produced by the more sensitive analyzer utilizing the fluorescamine reaction to detect the amino acids compared favorably with the chromatogram produced by the commercial analyzer utilizing the ninhydrin reaction with the exception that the proline peak was missing. Proline and hydroxyproline fail to yield fluorescence on reaction with fluorescamine unless converted from imines to primary amines.     

Hydroxyproline in Chiorella and its Metabolism in Tobacco Leaves

Hydroxyproline and other compounds were labeled with C¹⁴ by Chlorella pyrenoidosa supplied with C¹⁴O₂ in the light. The hydroxyproline recovered from a hydrolysate of the algae was administered through the cut bases of tobacco leaves. The leaves formed little proline from the hydroxyproline, but the C¹⁴ label was transferred to a variety of other amino acids. Although hydroxyproline is not abundant in plants, it appears to be an active metabolite.     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.