Amplified and selective assay of collagens by enzymatic and fluorescent reactions

Sensitive and selective assay of collagen is of substantial importance to the diagnostic study of health- and aging-related failures. In this paper, we describe a highly specific and sensitive method for the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4- dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA reagent can selectively detectN-terminal Gly-containing peptides (NGPs) in the presence of sodium borate and NaIO4. Under conditions optimized, this assay format for collagen, termed 3,4-DHPAA assay method showed a good linear relationship between the amplified FL signals and the collagen concentrations from 0.18 to 12 μg/ml. Therefore the sensitive determination of intracellular collagens in cheek tissue and HeLa cells was individually possible without any separation protocol. The dual recognitions of the collagens in the samples could be performed by the enzymatic digestion and the FL reaction. The proposed assay method enables the determination facile, specific, sensitive and quantitative for biogenic collagens

Prince Cangrande\u2019s Collagen: Study of Protein Modification on the Mummy of the Lord of Verona, Italy (1291\u20131329 AD)

The natural mummy of prince Cangrande, Lord of Verona, Italy (1291\u20131329 AD) was studied. Two samples were taken: rib bone and muscle. These samples were cleaved with trypsin and analysed by liquid chromatographic methods coupled to mass spectrometry (Q-TOF,ion-trap). Special attention was devoted to nonenzymatic protein modification\u2013\u2013the deamidation of asparagine and glutamine. A huge amount of collagen was determined in the tissues of the mummy (covering over 80 % of the sequence)\u2013\u2013collagen type I was identified in the rib bone and collagen types I and III in the muscle. A high overallpercentage of asparaginyl and glutaminyl residues were deamidated (up to 92 %). In agreement with the literature we can suppose that the deamidation of really old samples (at least 100-years-old) is mainly dependent on the burial conditions and/or thermal age and cannot serve as a precise \u201cmolecular clock\u201d