Hydroxyproline assay kit provides a convenient colorimetric method for the detection of total hydroxyproline from tissue, plasma, serum, or urine acidhydrolysates. Hydroxyproline is produced by hydroxylation of the amino acid proline by the enzyme prolyl hydroxylase following protein synthesis as a posttranslational modification. Quickzyme hydroxyproline assay this assay is similar to the total collagen assay, with the difference that no protocols and materials are included for collagen hydrolysis and instead of a collagen standard a hydroxyproline. Louis, mo and assayed by measuring the optical density at 555562 nm. Chloraminet was used to oxidize the free hydroxyproline for the production of a pyrrole.
Hydroxyproline assay kit perchloratefree, biovision, inc. Enzymatic method for determining hydroxyproline sigmaaldrich. Hydroxyproline 4hydroxy2pyrrolidinecarboxylic acid, a heterocyclic amino acid in terms of chemical structure, an imino acid. A modified assay for the determination of hydroxyproline in tissue is presented. It is an easy, convenient method which results in a chromogen with an absorbance maximum at. The quickzyme hydroxyproline assay kit is the only hydroxyproline based assay which does not need this laborious step, resulting in a fast assay. It has a physiological function in stabilizing the collagen 3d structure. Collagen analysis in fish tissue using the total collagen. Hydroxyproline assay kit colorimetric ab222941 provides a quick.
A high value for the hydroxyproline levels urine test depends on the age of an individual, and is as follows. Hydroxyproline and proline play key roles for collagen stability. The assay relies on the fact that the collagen triple helix is one of the few proteins that contain the amino acid hydroxyproline. They permit the sharp twisting of the collagen helix.
Its two isomeric forms trans4hydroxylproline and trans3hydroxylproline play a. This measurement is preferable to free serum hydroxyproline measurement in this setting. Spot urine testing is measurement of choice in screening for bony involvement by. Hydroxyproline reacts with erlichs reagent to give a red color that can be measured in a 96well plate reader capable of absorbance measurement at 550 nm. Suitability suitable for hydroxyproline detection in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples. For the assay of one unknown solution, nine dry 18 x 150 mm. Modified assay for determination of hydroxyproline in a tissue hydrolyzate. Hydroxyproline 4hydroxyproline, hyp is a common nonproteinogenic amino acid.
Hydroxyproline is a nonessential amino acid derivative, which can be produced in your body from the amino acid proline, so you do not need to get it from foods in order to be healthy 1. Lumistor l hydroxyproline offers a real alternative for those who wish to avoid animalbased collagen supplements. It is found only in collagen and elastin in mammals but exists in a number of other proteins in plants. Bone resorption is the major contributor to collagen turnover, and the constituent molecules, including hydroxyproline, are useful as markers of the rate of bone matrix resorption. This particular tissue sample is a purified bovine tendon collagen calbiochem which requires serial dilutions in order for the hydroxyproline concentration to be within the assay sensitivity range. Standard addition curve for determination of the hydroxyproline concentration of a tissue hydrolyzate. Although it is not directly incorporated into proteins, hydroxyproline comprises roughly 4% of all amino acids found.
Optimal assay conditions were determined using tissue homog enate and purified acid soluble collagen along with standard hydroxyproline. It is used to diagnose bone disorders such as pagets disease of the bone. A simplified method for the analysis of hydroxyproline in biological. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. The nterminal glycine reacts with the dye reagent forming a fluorescent complex. Hydroxyproline is found almost exclusively in collagen and measured hydroxyproline levels correlate to the levels of collagen present in the sample, which can indicate diseases involving collagen breakdown.
The whole lung tissue and one half the volume of the homogenized samples value later utilized in equation 1. Hydroxyproline colorimetric assay kit video protocol. Collagen content from hydroxyproline sample preparation 1. Defects in collagen synthesis lead to easy bruising, internal bleeding. Determination of hydroxyproline by a modified bergman and loxley rapid procedure reagent 1. Chloraminet was used to oxidize the free, hydroxyproline for the production of a pyrrole. Small amounts of urinary hydroxyproline are present as free amino acids. Provided with the required supplies and reagents for any preferred method, enzyme kinetics and inhibition studies can be successfully completed. Applications, estimation of hydroxyproline concentration in various biological samples. Hey labrats, having some issues with a hydroxyproline assay specifically the mak008 from sigmaaldrich. However, the magnitude of the increase in collagen between control and fibrotic lungs was markedly lower in this assay 25% increase compared to the hydroxyproline assay 300% increase. Hydroxyproline metabolism journal of biological chemistry. The amino acid hydroxyproline is synthesized through the posttranslational modification of proline by prolyl hydroxylase. K226100 hydroxyproline assay kit perchloratefree v3 biovision.
C onversion of proline to collagen hydroxyproline in a cell. Hydroxyproline, an amino acid formed upon hydrolysis of connectivetissue proteins such as collagen about 14 percent by weight and elastin but rarely from other proteins. Nov 10, 2017 this video demonstrates how to quantify hydroxyproline levels in biological samples using a simple, highly sensitive, addincubateread protocol k555. Small amounts of urinary hydroxy proline are present as free. After homogenization of both lungs, the homogenate samples were centrifuged with only saltsoluble collagens remaining. The most widely used collagen assay is based on hydrolysis of collagen to free amino acids, followed by measuring collagen specific hydroxyproline, using either hplc or a colorimetric method. Hydroxyproline article about hydroxyproline by the free. Thus, bacterial extracts consume l hydroxyproline via an initial epim.
This particular tissue sample is a purified bovine tendon collagen calbiochem which requires serial dilutions in order for the hydroxyproline concentration to be within the assay. The determination of hydroxyproline there has been a. Hydroxyproline, a major component of collagen, makes up about. The hydroxyproline assay has been previously described 2, 4, 18, 19. It is also used to monitor the treatment for bone disorders. For research use only not human or therapeutic use. Allol hydroxyproline was discovered in free form in sandalwood and is a component of toxic peptides in the deathcap. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Hydroxyproline 2s,4r4 hydroxyproline, hyp is a nonproteinogenic amino acid formed by the enzyme prolyl4hydroxylase, which catalizes the posttranslational modification of polypeptide proline residues following protein synthesis. The enzyme catalyzed reaction takes place in the lumen of the endoplasmic reticulum. Then, a chloramine t mixture is added to convert the hydroxyproline to a pyrrole. Our hydroxyproline assay kit is a quantitative colorimetric assay. For uncooked samples, place 6 g of sample in weigh pan, record weights and heat in 110 c oven for 1.
Cell biolabs hydroxyproline assay kit provides a convenient colorimetric method for the detection. Read the entire protocol before performing the assay. The classical hydroxyproline assay protocol is based on the oxidation of hydroxyproline to a pyrrole intermediate followed by reaction with ehrlichs reagent dissolved in concentrated perchloric acid. A novel method for accurate collagen and biochemical. The colourimetric assay described here quantifies free hydroxyproline e. K555100 hydroxyproline colorimetric assay kit biovision. This assay protocol is suitable for the colorimetric detection of hydroxyproline in cell and tissue culture supernatants, urine, plasma, serum, and other biological samples using the hydroxyproline assay kit mak008. Hydroxyproline is necessary for the construction of the bodys major structural protein, collagen. Hydroxyproline is a nonessential amino acid found in collagen and few other extracellular animal proteins. Hydroxyproline is formed only as a posttranslational modification in the peptide chain and proline hydroxylase does not hydroxylate free proline. Collagen per protein analysis in fresh, frozen and formalin. The formation of lhydroxyproline in living cells is effected by hydroxylation of the proline bonded to the protein components the o atom of the hydroxyl group is incorporated into hydroxyproline upon fixation of molecular o 2.
Hydroxyproline definition of hydroxyproline by medical. Hydroxyproline assay kit perchloratefree clinisciences. The modifications greatly reduce the time required for analysis of excised tissue as first introduced by stegemann and stalder 1. Because urinary hydroxyproline is derived almost entirely from collagen, it reflects the rate of collagen catabolism. May 26, 2019 what is the significance of the hydroxyproline levels urine test result. L hydroxyproline c5h9no3 cid 54196981 structure, chemical names, physical and chemical properties, classification, patents, literature, biological activities. We describe a high performance liquid chromatography hplc technique for separating and quantitating hydroxyproline in calvarial cultures. Easy assay for analysis of hydroxyproline quickzyme. Recovery of added standards ranged from 89 to 103% and intraassay. Hydroxyproline is formed only as a posttranslational modification in the peptide chain and proline hydroxylase does not hydroxylate free. The bac terial pathway from hydroxyproline to glutamate is quite dis tinct, however, from the reactions so far outlined in the utiliza tion of l hydroxyproline by mammalian enzymes 8. Serum free proline and free hydroxyproline in patients. Cell biolabs hydroxyproline assay kit provides a convenient colorimetric method for the detection of total hydroxyproline from tissue, plasma, serum, or urine acidhydrolysates.
First, the unknown samples or hydroxyproline standards are added to a 96 well plate. This kit does not use pca to detect and quantify hydroxyprolineeither measuring substrate consumption or overall production, the enzyme assays are used to monitor enzymatic activity. Hydroxyproline in plasma appears as both peptide bound and free forms. We use bd microtainer with a gel separator to get the serum from whole blood. Hydroxyproline definition of hydroxyproline by the free. Hydroxyproline is an amino acid present in appreciable quantities in collagen and excreted in the urine after collagen breakdown. While continuous assay sampling methods give activity readings.
Although serum hydroxyproline and urinary hydroxyproline measurements are strongly correlated, urinary hydroxyproline measurement in 24hour or spot urine is easier to collect. Hydroxyproline assay kit perchloratefree, biovision. Analysis of hydroxyproline by high performance liquid. Small amounts of urinary hydroxy proline are present as free amino acids. Hydroxyproline assay kit has been used to determine the concentration of hydroxyproline and collagen. Commence assay to each tube add sircol dye reagent 1. Hydroxyproline is a major component of the protein collagen, comprising roughly. First isolated 1902 from gelatin, a breakdown product of collagen, hydroxyproline is one of several socalled nonessential. Hydroxyproline was first isolated from gelatin hydrolyzates in 1902. Recovery of added standards ranged from 89 to 103% and. Bioassay systems collagen assay kit ecol100 quantifies collagen in samples. Assay preparation of assay reagents the following 2 assay reagents are stable for 23 hours after preparation, and should be prepared after sample preparation, just prior. It is a quick and convenient protocol, yielding a brightlycolored chromophore with an absorbance peak at 560 nm.
Hydroxyproline determination in serum and gingival. A simplified method for the analysis of hydroxyproline in. Genways hydroxyproline assay kit is designed to measure. It can be used to measure hydroxyproline from other biological samples such as serum or urine if they have undergone a prior purification process. The second part of this chapter describes the use of sodium dodecyl sulfate sds polyacrylamide gel electrophoresis page to isolate, detect and quantify changes in the sol.
Meanwhile, free hydroxyproline and ammonia nitrogen contents increased by 81. Our hydroxyproline assay kit is a quantitative colorimetric assay for measuring the hydroxyproline concentration in protein samples. The hydroxyproline levels urine test helps determine the level of hydroxyproline in urine. Perchloric acid is a hazardous material that is both toxic and highly reactive, requiring special handling and wastedisposal protocols. The addition of ehrlichs reagent resulted in the formation of a chromophore that can be measured at 550 nm.
A hydroxyproline standard sample also from the same company is used to create a standard curve. R4hydroxyproline, hyp is a nonproteinogenic amino acid formed by the enzyme prolyl4hydroxylase, which catalizes the posttranslational modification of polypeptide proline residues following protein synthesis. Hydroxyproline 4 hydroxyproline, hyp is a common nonproteinogenic amino acid. Hydroxyproline colorimetric assay kit, biovision vwr. The assay is applicable for all types of samples, including tissue. Louis, mo or thermofisher waltam, ma unless otherwise noted. Request pdf a modified hydroxyproline assay based on hydrochloric acid.
The addition of ehrlichs reagent resulted in the formation of a chromophon. Collagen per protein analysis in fresh, frozen and. Comparison of collagen content in skin wounds evaluated by. Hydroxyproline 4 hydroxyproline is a common nonproteinogenic amino acid. Hydroxyproline assay kit sufficient for 100 colorimetric. Hydroxyproline assay kit is designed to measure hydroxyproline in tissue or proteinpeptide hydrolysates. Biovisions hydroxyproline assay kit perchloratefree uses a proprietary acidic developer solution to accurately measure hyp in tissue and proteinpeptidase hydrolysates without the use of hazardous perchlorates. Patented l hydroxyproline is a primary constituent of skin collagen, connective tissue and bone. The two assay methods show excellent correlation r2 0. Hydroxyproline is a nonessential amino acid, which means that it is manufactured from other amino acids in the liver.
It measures the total amount of amino acids present in the hydrolyzed sample, with exception of proline and hydroxyproline. Hydroxyproline as a biomarker in liver disease 17 the excretion of hydroxyproline in serum and urine promotes the easy diagno sis of the severity of hepatic. Hydroxyproline content is quantified these days by erlichs reagent sigma chemical co. Hydroxyproline assay kit works for quantitation of the total collagen of any types and species in tissue specimens and tissue homogenates. Featuring a simple and quick protocol, this hydroxyproline assay yields a brightlycolored chromophore with an absorbance peak at 560 nm. Hydroxyproline benefits, deficiency, hyperhydroxyprolinemia. First chemosensor for selective detection and quantification.
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