Amplified and selective assay of collagens by enzymatic and fluorescent reactions

Yasmin, Hasina; Kabashima, Tsutomu; Rahman, Mohammed Shafikur; Shibata, Takayuki; Kai, Masahiro

doi:10.1038/srep04950

Cited by 16 publications

(16 citation statements)

References 42 publications

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“…More recently, a method was described for quantifying collagen in collagenase-digested tissue based on binding of a fluorescent molecule to peptides containing N-terminyl glycine residues. 21 Each of these biochemical methods for measuring collagen exploits a different, unique attribute of collagen's amino acid sequence or structure.…”

Section: Introductionmentioning

confidence: 99%

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content

Cissell

Link

et al. 2017

Tissue Engineering Part C: Methods

163

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Collagen quantification has long been relevant to biomedical research and clinical practice to characterize tissues and determine disease states. The hydroxyproline assay, while a broadly employed method of quantifying collagen, uses perchloric acid to dissolve Ehrlich's reagent. Since perchloric acid poses occupational safety hazards and high costs, in this study, a new hydroxyproline assay was developed that replaces perchloric acid with a relatively safer and cheaper alternative, hydrochloric acid (HCl). To validate this biochemical technique, first, using either acid to dissolve Ehrlich's reagent, the assays were completed for native and engineered collagenous tissues. No statistical differences were identified between the assays (p = 0.32). Subsequently, both biochemical techniques were compared to amino acid analysis, considered a proteomics gold standard. Interestingly, utilizing HCl in lieu of perchloric acid yielded greater concordance with amino acid analysis (ρ = 0.980) than did the traditional assay (ρ = 0.947); that is, the HCl-based assay more closely estimates hydroxyproline content, and, consequently, true collagen content. Thus, using Ehrlich's reagent containing HCl in the hydroxyproline assay represents an advance in both mitigating laboratory safety hazards and improving biochemical collagen quantification.

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Section: Introductionmentioning

confidence: 99%

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content

Cissell

Link

et al. 2017

Tissue Engineering Part C: Methods

163

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“…The bacterial collagenase cleaves collagens at the N-terminal of the Gly residue [28] [29]. Therefore, abundant N-terminal Gly-containing oligopeptides are produced from one molecule of collagen [24]. As shown in Scheme 1, human collagenase generally cleaves collagens at only Gly-Leu or Gly-Ile binding sites, thus producing large peptide fragments [2].…”

Section: Principle Of Collagen Substrate Assaymentioning

confidence: 99%

“…Previously [24], we reported sensitive quantification of human collagens by using the fluorogenic reagent, DHPAA for labeling N-terminal Gly-containing oligopeptides after enzymatic digestion by bacterial collagenase. Here, we used DHPAA to sensitively detect the activity of human collagenase because the enzyme cleaves collagens into large fragments.…”

Section: Introductionmentioning

confidence: 99%

“…Other selective FL reagents include glyoxal for Nterminal Trp-containing peptides [16], 1,2-dihydroxybenzene(DHB) and borate for N-terminal Phe-, Ile-, Leu-, Val-, or Ala-containing peptides [17], DHB and 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid buffer at pH 7.5 for N-terminal Ser-containing peptides [18], 3,4-dihydroxyphenylacetic acid (DHPAA) for N-terminal Gly-containing peptides [19], and 3,4-dihydroxybenzoic acid for N-terminal Pro-containing peptides [20]. These reagents could be used to analyze several peptides in complex mixtures such as tissues and enzymatic digests [18] [21]- [24].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates

Ejupi¹,

Dragusha²,

Kabashima³

et al. 2015

AER

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A selective, sensitive, and convenient assay for human collagenase is required because of its implication in diseases such as rheumatoid arthritis, osteoarthritis, and tumors. Here, a novel assay for human collagenase activity is described in which enzymatic degradation of native collagen or acetyl peptide is determined by using a fluorogenic reaction for oligopeptides. The oligopeptides are quantified spectrofluorometrically with either 3,4-dihydroxyphenylacetic acid or 1,2-dihydroxybenzen reaction in the presence of sodium periodate and sodium borate (pH 7 -8). These reactions can selectively convert N-terminal Gly-containing oligopeptides and N-terminal Ile-containing oligopeptides to fluorescence (FL) compounds, respectively, but not proteins, acetyl peptides or amino acids. Under optimized conditions using 1.65 μM collagen IV or 1.5 mM Ac-GPQGI-AGQ as substrates, this assay exhibits a proportional relationship between FL intensities and human collagenase-3 (MMP-13) concentrations. It can assay endogenous collagenase activities in several biological samples, such as cultured human cells and cheek tissue.

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“…These non-FL reagents are 1) hydroxylamine, cobalt(II) and borate for N -terminal Tyr-containing peptides 31 , 2) glyoxal for N -terminal Trp-containing peptides 32 , 3) catechol (1,2-dihydroxybenzene) , NaIO 4 and borate for N-terminal Phe-, Leu-, Val-, or Ala-containing peptides 33 , 4) catechol, NaIO 4 and 2-[4-(2-hydroxyethyl) piperazin-1-yl] ethanesulfonic acid (HEPES) buffer (pH 7.5) for N -terminal Ser-containing peptides 34 , 5) 3,4-dihydroxyphenylacetic acid, NaIO 4 and borate for N -terminal Gly-containing peptides 35 and 6) 3,4-dihydroxybenzoic acid, NaIO 4 and borate for N -terminal Pro-containing peptides 36 . Those reagents were applied to analyze several peptides in complex mixtures such as tissues and enzymatic digests, and also to selectively assay various enzyme activities 37 38 39 40 . Hence, a limited number of amino acids in peptides can be selectively converted to individual FL compounds for the sensitive assay of particular enzymes.…”

mentioning

confidence: 99%

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

Zhu

Kabashima

et al. 2015

Sci Rep

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Convenient drug-resistance testing of viral mutants is indispensable to effective treatment of viral infection. We developed a novel fluorometric assay for phenotypic differentiation of drug-resistant mutants of human immunodeficiency virus-I protease (HIV-PR) which uses enzymatic and peptide-specific fluorescence (FL) reactions and high-performance liquid chromatography (HPLC) of three HIV-PR substrates. This assay protocol enables use of non-purified enzyme sources and multiple substrates for the enzymatic reaction. In this study, susceptibility of HIV mutations to drugs was evaluated by selective formation of three FL products after the enzymatic HIV-PR reaction. This proof-of-concept study indicates that the present HPLC-FL method could be an alternative to current phenotypic assays for the evaluation of HIV drug resistance.

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Amplified and selective assay of collagens by enzymatic and fluorescent reactions

Cited by 16 publications

References 42 publications

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content

A Modified Hydroxyproline Assay Based on Hydrochloric Acid in Ehrlich's Solution Accurately Measures Tissue Collagen Content

Spectrofluorometric Assays of Human Collagenase Activity Using Native Collagen and Acetyl-Peptide Substrates

Fluorometric assay for phenotypic differentiation of drug-resistant HIV mutants

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