Hyaluronic acid, in its hydrated form, is a unique polysaccharide polymer, often referred to as a 'gentle giant'. It is found in many tissues, but particularly in cartilage and synovial fluid and consists of a lengthy, flexible, non-branching chain with a repeating disaccharide pattern. This pattern comprises alternating uronic acid and aminosugar units. Hyaluronic acid is involved in a wide range of roles including tissue hydration and lubrication, cell signalling, extracellular matrix reorganisation and wound repair.
This involvement of Hyaluronic acid in tissue lubrication and wound healing has meant it has become a well-known additive to both medical and cosmetic skincare treatments, as well as treatments for osteoarthritis. The ability to assay or measure hyaluronic acid is therefore of interest to scientific researchers from a range of disciplines.
Discovering the J-Aggregate Effect in Cyanine DyesIn 1936, Edwin Jelley made a fascinating observation, documented it in a letter to Nature (Nature 138, 1009 - 1010). He noted a peculiar behaviour of certain cyanine dyes, that when dissolved in 5 M NaCl, they dyes exhibited a third absorbance peak at a longer wavelength, around 650nm. In deionized water, however, they displayed only a double peak at approximately 540nm and 570nm. The 650nm peak in concentrated dye solutions resulted from the aggregation of dye molecules and was later termed a 'J-aggregate,' in honor of Edwin Jelley. The J-aggregate is known as a supra-molecular complex, formed by stacking individual dye molecules.
Subsequent research in the 1960s, notably by Kay et al. (J. Physical Chem. 68, 1896 – 1906), revealed that various biological polymers, including proteins, DNA, polar lipids, and glycosaminoglycans, could also induce this third absorbance peak. This phenomenon led to the development of the Purple-Jelley assay, named after the purple color of the dye reagent and Edwin Jelley himself.
During the assay, hyaluronic acid is selectively purified during the assay sample preparation protocol. This is then reacted with the Purple-Jelley dye reagent, and the absorption of the characteristic third wavelength recorded. By comparison with a calibration curve the hyaluronic acid content of the sample can be measured.
Step 1. The assay protocol takes tissue samples through a sequential sample preparation protocol which involves enzymatic protein digestion, followed by precipitation and purification of GAGs, culminating in the precipitation of purified Hyaluronic acid.
Step2. The processed sample is then incubated for 10 minutes with the Purple-Jelley dye reagent, forming a coloured product which can be measured spectrophotometrically.
Step 3. The Hyaluronic acid content of unknown samples can be calculated by comparison against a calibration curve prepared using a standard comprising hyaluronic acid (supplied with the kit).
A list of suggested sample types can be found under the 'Assay Specification' tab.
In-vivo: Hyaluronic acid purified from in-vivo tissues. The kit protocol involves extraction and purification of hyaluronic acid prior to reaction with the Purple-Dye reagent.
This kit is designed for research use only. Not for use in diagnostic procedures.
Kit requires access to a centrifuge, as well as a spectrophotometer/colorimeter capable of colorimetric, absorbance detection at 655nm.
Specific sample preparation protocols may require customer to provide further reagents, consult assay manual for further information.
1. Purple-Jelley Dye Reagent (1x 20ml)
2. Hyaluronan Reference Standard (1x 5ml, 0.2mg/ml soluble Hyaluronic Acid)
3. Precipitating Reagent (2x 34ml)
4. Sodium Chloride (1x 20ml)
5. Cetylpyridinium Chloride (1x 20ml)
6. TRIS-buffered Saline (5x tablets)
7. 2ml screw-cap tubes for preparation of samples.
8. Assay kit manual
NB: Additional reagents may be required for sample preparation prior to assay. Consult manual or contact us for further details.
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