Collagen is the most abundant protein in mammals, accounting for nearly 30% of body protein content. Collagen is an extracellular scaffolding protein found in skin, tendons, bone, cartilage, ligaments and muscles, amongst other tissues. There are more than twenty different characterized types of collagen, but the fibrilous types I, II and III comprise more than 90% of the total collagen in mammals. Collagen has a triple helical structure, consisting of three ‘preprocollagen' strands with a repeating Gly-X-Hyp tripeptide sequence, where X is any amino acid and Hyp is hydroxyproline, a post-translationally modified amino acid formed from proline by the enzyme prolyl-4-hydroxylase. In animals, hydroxyproline is found almost entirely in collagen (which contains approximately 12-14% hydroxyproline by mass) and contributes to its structural rigidity and high tensile strength. Hydroxyproline is thus a direct measure of the amount of collagen present in tissues. Many diseases are believed to affect collagen turnover, including tumor invasion and metastasis, rheumatoid arthritis, cardiopulmonary fibrosis and muscular dystrophy. Assay Genie's Total Collagen Assay Kit II (Perchlorate-Free) is a quick and convenient protocol for detection of small amounts of collagen in a variety of biological samples. The assay is based upon hydrolysis of samples to yield free Hyp, which is oxidized and reacted to give a brightly-colored chromophore with an absorbance peak at 560 nm. The assay employs a proprietary acidic developer solution to accurately measure collagen in hydrolysates without the use of hazardous perchloric acid, obviating the need for any special handling and waste-disposal protocols. The assay can detect a minimum of 0.5 µg collagen per well in a 96-well format and generates colorimetric results that are virtually identical to those of the classical perchloric acid-based method

Figure: (a) Hydrolyzed Collagen I Standard curve. (b) Correlation between collagen standard curve absorbance values obtained using the classical perchloric acid-based assay kit and the perchlorate-free assay. The two assay methods show excellent correlation (R 2 > 0.999). (c) Estimation of total collagen content in rat tissues. Rat leg muscle, spleen, heart and lung samples were homogenized with dH₂O hydrolyzed with 10N NaOH for 1 hour at 120°C and neutralized with 10N HCl. For each sample, 10 μl of the final hydrolysate was assayed. Collagen levels (calculated as μg collagen/mg wet tissue) for the samples were: 13.80 ± 0.73 μg/mg for muscle, 3.77 ± 0.54 μg/mg for spleen, 4.55 ± 0.81 μg/mg for heart and 12.51 ± 1.27 μg/mg for lung. Data are mean ± SEM of 3-4 replicates.