High-throughput quantification of hydroxyproline for determination of collagen

An accurate and high-throughput assay for collagen is essential for collagen research and development of collagen products. Hydroxyproline is routinely assayed to provide a measurement for collagen quantification. The time required for sample preparation using acid hydrolysis and neutralization prior to assay is what limits the current method for determining hydroxyproline. This work describes the conditions of alkali hydrolysis that, when combined with the colorimetric assay defined by Woessner, provide a high-throughput, accurate method for the measurement of hydroxyproline.     

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.     

Intracellular location of triple helix formation of collagen. Enzyme probe studies.

Primary cultures of chick embryo fibroblasts were used to study ribosomal events in the processing of procollagen. Polyribosomes from radiolabeled cells were subjected to enzyme probe analysis using collagenase and pepsin digestion to assess both the amount of procollagen present on the polyribosomes and the conformation of the molecule. The peptides rendered dialyzable by each enzyme treatment were analyzed for radioactive proline and hydroxyproline. Approximately 30% of the nascent proteins were collagenous. Although some hydroxyproline was dialyzable in the pepsin-treated material, a low ratio of hydroxyproline to proline (0.04) indicated that considerable amounts of noncollagenous proteins were digested. Polyribosomal material, previously treated with pepsin, was digested with purified collagenase. Similarly, collagenase-digested polyribosomes were treated with pepsin. The pepsin pretreatment released noncollagenous protein and served to purify the remaining ribosomally bound pepsin-resistant collagenous protein. Collagenase treatment of the pepsin-resistant ribosomally bound peptides released peptides with a hydroxyproline to proline ratio of 0.65, indicating that considerable hydroxylation of proline occurs on nascent ribosomally bound procollagen. This finding combined with the well documented stabilizing effect of hydroxyproline on the collagen triple helix and the demonstrated resistance of ribosomally bound procollagen to pepsin digestion indicates that the collagen triple helix may well form on the polyribosome.     

Characterization of the skin collagen of a surface water fish Scomberoides commersonianus.

The molecular organization of the skin collagen in the fish S. commersonianus has been investigated. The contents of imino acids proline and hydroxyproline are less in the skin collagen. The major collagenous component of fish skin is homologous to type I collagen. The number of CNBr peptides in fish skin collagen alpha 1 chains is two times that of rat skin alpha 1 CNBr peptides. The proline-hydroxyproline ratio in the six peptides studied was 1.66-3.5 as compared to that of rat skin collagen (0.67-1.94). This indicates that proline and hydroxyproline are not uniformly distributed in the collagen molecule in fish skin collagen.     

Application of high-pressure liquid chromatography to studies of collagen production by isolated cells in culture

Techniques for assessing collagen production by cells in culture are usually based on evaluation of uptake of radiolabeled proline into collagen. Although simple in theory, this approach is often flawed because of uncertainties concerning the specific activity of labeled proline in the precursor pool for collagen synthesis. An alternative approach is to assess collagen production directly by measuring hydroxyproline in proteins secreted by cultured cells, although this has been difficult, due to the insensitivity of the methods available. Here we apply high-pressure liquid chromatography using reverse-phase elution of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole derivatives of hydroxyproline to measure collagen production by fibroblasts. The method is easy to perform and allows quantitation of hydroxyproline down to 5 pmol, making it applicable to fibroblasts in 12-well culture plates. Collagen production was shown to be constant over a period of 24 h, with a mean rate of 391 +/- 18 (SE n = 14) ng collagen/10(6) cells/h. Similar values were obtained using thin-layer chromatography and an enzyme-linked immunosorbent assay for type I collagen, but these techniques were judged to be less convenient and required additional assumptions compared with the technique described here in full.     

Self-association of collagen triple helic peptides into higher order structures

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as beta-amyloid formation. The triple helical form of (Pro-Hyp-Gly)(10), a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleation-growth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25 degrees C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (Pro-Hyp-Gly)(10) forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation

Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used as an experimental model to provide insights into the role of hydroxyproline in molecular folding and fibril formation. By using chemically cross-linked molecules, we show here that the absence of hydroxyproline residues does not prevent correct folding of the recombinant collagen although it markedly slows down the propagation rate compared with bovine fully hydroxylated homotrimeric collagen I. Relatively slow cis-trans-isomerization in the absence of hydroxyproline likely represents the rate-limiting factor in the propagation of the unhydroxylated collagen helix. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility as well as a reduced melting temperature compared with native homotrimers and heterotrimers, whereas the distribution of charged amino acids was unchanged. However, unlike with bovine collagen I, the recombinant collagen did not self-assemble into banded fibrils in physiological ionic strength buffer at 20 degrees C. Striated fibrils were only obtained with low ionic strength buffer. We propose that, under physiological ionic strength conditions, the hydroxyl groups in the native molecule retain water more efficiently thus favoring correct fibril formation. The importance of hydroxyproline in collagen self-assembly suggested by others from the crystal structures of collagen model peptides is thus confirmed experimentally on the entire collagen molecule.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs

1. After the administration of labelled proline to guinea pigs deprived of ascorbic acid for 15 days, the dorsal skin was examined 5 days later in an attempt to detect the presence of hydroxyproline-deficient collagen (protocollagen). The extent of incorporation of proline into skin collagens indicated a severe impairment of collagen synthesis. 2. A comparison of proline and hydroxyproline specific radioactivities in diffusible peptides obtained by treatment with collagenase of either purified skin collagens or direct hot-trichloroacetic acid extracts of skin failed to indicate the presence of protocollagen. Possible reasons for this are discussed. 3. The incorporation results did not indicate an inability of normal collagen, i.e. collagen hydroxylated to the normal degree, to cross-link in scurvy. 4. Incorporation of labelled proline into aortic elastin isolated from the same animals did not indicate a decrease in elastin biosynthesis in ascorbic acid deficiency, beyond that attributable to the inanition accompanying the vitamin deficiency. The proline/hydroxyproline specific-radioactivity ratio in elastin from scorbutic guinea pigs was about 6:1 in contrast with the 1:1 ratio in control groups. It is concluded that the formation of elastin hydroxyproline was ascorbate-dependent and that a hydroxyproline-deficient elastin is formed and retained in scurvy. The formation of desmosines was unimpaired in scorbutic animals. 5. Studies with chick embryos confirmed the formation of elastin hydroxyproline from free proline. Incorporation of free hydroxyproline into elastin hydroxyproline was negligible. 6. Digestion of solubilized samples with collagenase indicated that the hydroxyproline in guinea-pig aortic elastin preparations was not derived from contamination by collagen. It is suggested that most if not all of the hydroxyproline in the guinea pig elastin preparations investigated can be considered an integral part of the elastin molecule.     

Prediction of collagen stability from amino acid sequence

An algorithm was derived to relate the amino acid sequence of a collagen triple helix to its thermal stability. This calculation is based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm. However, predicted T(m) values are significantly higher than experimental values when there are strings of oppositely charged residues or concentrations of like charges near the terminus. Application of the algorithm to collagen sequences highlights regions of unusually high or low stability, and these regions often correlate with biologically significant features. The prediction of stability from sequence indicates an understanding of the major forces maintaining this protein motif. The use of highly favorable KGE and KGD sequences is seen to complement the stabilizing effects of imino acids in modulating stability and may become dominant in the collagenous domains of bacterial proteins that lack hydroxyproline. The effect of single amino acid mutations in the X and Y positions can be evaluated with this algorithm. An interactive collagen stability calculator based on this algorithm is available online.     

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.     

胶原海绵的羟脯氨酸含量测定

选用普通实验室均能实现的 Woessner第 法对自制胶原海绵和 Gelfix(国外样品 )进行了羟脯氨酸含量测定 ,结果表明 ,该方法操作简单 ,重复性好 :自制胶原海绵的羟脯氨酸含量稳定 ,与国外样品的羟脯氨酸含量接近 ,且与胶原蛋白的羟脯氨酸含量接近 ,证实了两种胶原海绵的纯度均较高     

Characterization of high affinity binding motifs for the discoidin domain receptor DDR2 in collagen

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that are activated by native triple-helical collagen. Here we have located three specific DDR2 binding sites by screening the entire triple-helical domain of collagen II, using the Collagen II Toolkit, a set of overlapping triple-helical peptides. The peptide sequence that bound DDR2 with highest affinity interestingly contained the sequence for the high affinity binding site for von Willebrand factor in collagen III. Focusing on this sequence, we used a set of truncated and alanine-substituted peptides to characterize the sequence GVMGFO (O is hydroxyproline) as the minimal collagen sequence required for DDR2 binding. Based on a recent NMR analysis of the DDR2 collagen binding domain, we generated a model of the DDR2-collagen interaction that explains why a triple-helical conformation is required for binding. Triple-helical peptides comprising the DDR2 binding motif not only inhibited DDR2 binding to collagen II but also activated DDR2 transmembrane signaling. Thus, DDR2 activation may be effected by single triple-helices rather than fibrillar collagen.     

The structure of l-CB3, a cyanogen bromide fragment from the central portion of the l chain of rat collagen. The tryptic peptides from skin and dentin collagens

The mixture of peptides released by tryptic hydrolysis of the collagen CNBr peptide, αl-CB3, has been resolved by ion-exchange chromatography. The resultant eleven tryptic peptides ranged in size from 3 to 46 amino acids and accounted for all the amino acids of the parent CNBr peptide. Two of the lysines in αl-CB3 from rat dentin collagen were shown to be hydroxylated to a substantial degree by isolation of the appropriate hydroxylysine-containing tryptic peptides. An analysis of the tryptic peptides indicated that αl-CB3 from dentin collagen is identical in structure to that from skin collagen, if hydroxylysine and hydroxyproline are considered equivalent to lysine and proline, respectively.     

Regional differences in water content,collagen content,and collagen degradation in the cervix of nonpregnant cows

The cow could be a suitable model for studies concerning functional changes of the cervix. However, as in many species, the bovine cervix becomes softer in texture during the follicular phase of the estrous cycle compared to the luteal phase. In the present study, we explored if changes in the collagen network take place that could be responsible for this phenomenon and if regional differences in water content, collagen content, and collagen degradation along the cross-sectional and longitudinal axes of the cervix were present. Two groups of nonpregnant animals with different progesterone status were studied. One group (n = 11) was under high progesterone influence, and the other group (n = 12) was under low progesterone influence. The water content was derived from the weight of the samples before and after lyophilization. The collagen content and the ratio of collagenous to noncollagenous proteins (hydroxyproline:proline ratio) were determined by performing amino acid analysis on hydrolyzed samples using high-performance liquid chromatography. Collagen denaturation was quantified with a colorimetric assay by determining the amount of hydroxyproline released from samples treated with alpha-chymotrypsine. The water content of the superficial layer of the submucosa was always significantly (P < 0.01) higher than the water content of the deep layer in the vaginal, mid, and uterine segments, but this was unrelated to the progesterone status of the animals. No effect of the tissue layers or of the progesterone status of the animals on the collagen content was observed, but an effect of segment was noted. The collagen content (mug/mg dry wt) in the vaginal segment of the cervix was significantly higher than in the mid (P < 0.05) and the uterine (P < 0.01) segments. The hydroxyproline:proline ratio showed the same pattern as the collagen content. The percentage of collagen denaturation in the superficial layer was always significantly (P < 0.01) higher than that in the deep layer, but no effect of the progesterone status or of the segment along the longitudinal axis was seen. It is concluded that regional differences in collagen biochemistry are present in the cervix of nonpregnant cows, which may account for the difference in firmness of different parts along the circular or the longitudinal axis of the cervix. However, differences in texture of the cervix between the two groups of cows could not be explained by differences in the collagen content, percentage of collagen denaturation, or water content.     

Synthesis and physical properties of (hydroxyproline-proline-glycine)10: Hydroxyproline in the X-position decreases the melting temperature of the collagen triple helix

The collagen-like polytripeptide (hydroxyproline-proline-glycine)₁₀ was synthesized with a solid-phase procedure. Analytical ultracentrifugation indicated that the peptide in aqueous solution at 6 °C had a molecular weight of 2550, the expected size of a single chain. The peptide had a relatively small negative optical rotation at 578 nm, and it did not show a thermal transition as is seen with collagen or collagen-like polytripeptides which form triple helices. At low temperatures in aqueous solution, the circular dichroism spectrum was similar to that of triple-helical collagen and collagen-like peptides in that there was a positive peak at 224 nm and a negative peak at 200 nm. The amplitudes of the peaks, however, were considerably less than the peaks obtained with triple-helix proteins and peptides. Since (proline-proline-glycine)₁₀ was triple helical under the same conditions, the results demonstrated that hydroxyproline in the X-position of the repeating -glycine-X-Y- sequences decreases rather than increases, the thermal stability of the triple helix. This positional specificity cannot be explained by any of the current models for the structure of the triple helix or any of the current proposals for how hydroxyproline stabilizes the structure.     

Vitrosin: a member of the collagen class

Vitrosin, a fibrous protein obtained from the vitreous humor of the eye in the form of an indefinitely long fibril about 100 to 150 A in diameter, has been identified as a member of the collagen class of proteins. It is characterized by the collagen wide-angle x-ray diffraction pattern, and axial periodicity of about 640 A determined by electron microscopy and small-angle x-ray diffraction, an amino acid pattern characteristic of collagen as determined by paper chromatography, and a hydroxyproline and glycine content also typical of collagen. The glycine-hydroxyproline ratio is somewhat lower than that for most vertebrate collagens.     

Characterization of pepsin-solubilized bovine heart-valve collagen.

Collagens extracted from heart valves by using limited pepsin digestion were fractionated by differential salt precipitation. Collagen types were identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, amino acid analysis and cleavage with CNBr. Heart-valve collagen was heterogeneous in nature, consisting of a mixture of type-I and type-III collagens. The identity of type-III collagen was established on the basis of (a) insolubility in 1.7 M-NaC1 at neutral pH, (b) behaviour of this collagen fraction on gel electrophoresis under reducing and non-reducing conditions, (c) amino acid analysis showing a hydroxyproline/proline ratio greater than 1, and (d) profile of CNBr peptides on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showing a peak characteristic for type-III collagen containing peptides alpha1(III)CB8 and alpha1(III)CB3. In addition to types-I and -III collagen, a collagen polypeptide not previously described in heart valves was identified. This polypeptide represented approx. 30% of the collagen fraction precipitated at 4.0 M-NaCl, it migrated between beta- and alpha1-collagen chains on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and its electrophoretic behaviour was not affected by disulphide-bond reduction. All collagen fractions from the heart valves contained increased amounts of hydroxylysine when compared with type-I and -III collagens from other tissues. The presence of beta- and gamma-chains and higher aggregates in pepsin-solubilized collagen indicated that these collagens were highly cross-linked and suggested that some of these cross-links involved the triple-helical regions of the molecule. It is likely that the higher hydroxylysine content of heart-valve collagen is responsible for the high degree of intermolecular cross-linking and may be the result of an adaptive mechanism for the specialized function of these tissues.     

胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。     

Inhibition of collagen breakdown by diphenylhydantoin

Gingival tissue obtained from diphenylhydantoin-treated patients was cultured in the presence of [¹⁴C]proline for 24 h. The radioactive medium was removed and the tissue cultured for three days more. DNA, protein, hydroxyproline, proline and radioactivity determinations in the tissue indicated increased cellular proliferation, increased collagen contents and decreased breakdown of collagen in the affected tissues. The media were assayed for dialyzable and non-dialyzable hydroxyproline contents. It was found that the media in which diphenylhydantoin tissues were cultured contained more than twice as much non-dialyzable hydroxyproline than the controls. It was concluded that diphenylhydantoin brought about a reduction in collagen breakdown thus explaining the accumulation of hydroxylated collagen in the tissue.