We used to purify collagen II from chicken sternum cartilage, we use this protein to induce arthritis in CIA model. The purity of the protein is confirmed with SDS-PAGE, but this technique does note reveal contaminating proteoglycans. Is there a standard 230/260/280 nm absorption ratio to compare it to?
Thank you Dr Biao for the answer,
But I can't see how lyophilization and weight reveal contaminating proteoglycans or confirm the purity of the collagen II. Lyophilization can be used to quantify collagen, thing we do with modified Bradford reagent (with SDS 0,015 % final concentration, collagen precipitate in normal Bradford). There are different means to confirm the purity: RP-HPLC, determination of hydroxyproline content, determination of proteoglycans content. Du to technical limitations I can't use any of these three methods. Comparison with a standardized absorbance ratio of highly purified collagen II will be accessible alternative.
There is a very cheap and easy way to quantify collagen that is not well known. This is based on measuring primary amine content via fluorescamine assays, (or other primary amine assays) before and after digestion with micrococcal collagenase. The latter digests collagen into very short peptides, (dipeptides I believe), and so increases the number of primary amines hugely. This works best if you can eliminate other sources of primary amines by washing. The importance of the latter depends on the collagen levels you are examining. Try it on your pure collagen II samples first.
thanks for the answer but i need to confirm the purity not to quantify the collagen
Quite right, quantifying the collagen would only be one part of the answer. If you suspect proteoglycan contamination then this could be estimated via Dimethylene Blue assay which essentially measures chondroitin sulphate levels. The latter can be used to create a dose response curve to make sure that your samples are on the linear assay range. A ratio of the readings obtained here verses readings in the collagen assay suggested previously would give you an idea of purity. I think there are commercial DMB kits available for estimating chondroitin sulphate levels.
Thank you Jim Coote for your interest in my question. I have done researches about the DMMB method for proteoglycan dosage and it need DMMB (sigma: 341088) and chondroitin sulfate (sigma: C4384) as a standard. I am trying to get some of these two products.
But i still need the 230/260/280 nm absorption ratios of collagen II. because collagen II lambda max is 230 nm, the presence of any contaminating protein (lambda max 280 nm) will change this absorbance ratio. having a standardized absorbance ratio for highly purified collagen will give an easy way to monitor purification process and to confirm the purty of the final collagen II.
The extracted collagen may have other proteins (non-collagen protein). To verify this, I did maximum absorption of collagen, which is maximum at ~ 230-240 nm. In general, collagen has no or least tyrosin content (which could absorbs UV light at 280 nm and mainly present in non-collagen protein). If you sample does not exist any peak at 280 nm, which means pure..
thank you Jeevithan Elango for the answer.
i read your paper and the one of Kittiphat Tanabawon et al. and the two are interesting. i will do the absorption spectrum, but i still need to compare my results with those of other researchers. i will be thankful if you can upload the absorption spectrum of the collagen II you purified.
I use the methode of Griffiths et al. (2007): http://link.springer.com/protocol/10.1007/978-1-59745-402-5_15
Protein Concentration Method: CII does not absorb ultraviolet light at 280 (only 230 nm). Because of helical nature, native CII does not react with usual protein detection reagents. To measure CII protein concentrations, denature the CII by heating to 60°C for 10 min in water bath and use 0.015% sodium dodecyl sulfate (SDS) (final) with the Bradford Reagent.
For standard curve i use 2mg/ml BSA from sigma (poduct code: C4384).
Collagen purification protocol was adapted from the works of Rosloniec et al. (2001), Griffiths et al. (2007), Cao and Xu (2008) and Rosloniec et al. (2010) which is almost the same protocol consisting of five main steps:
1-the removal of proteoglycans by salt extraction.
2-digestion of cartilage with pepsin.
3-differential precipitation of pepsin resistant collagen II by NaCl and dialysis against sodium phosphate buffer.
4-adsorption of the remaining proteoglycans and pepsin on DEAE- sepharose gel.
5-salting out of pure collagen II and lyophilisation
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