Youare trying to come up with a simple colorimetric test for thedetection of hyp

Question

Youare trying to come up with a simple colorimetric test for thedetection of hypersulfated chondroitin sulfate as a contaminant ofheparin sulfate. You have access to

-samples of heparin and/or tainted heparin having equal potenciesas anti-coagulants

-heparinases, enzymes that are specific for the hydrolysis ofheparin sulfate

-copper reagents that turn yellow upon reacting with reducingsugars

Describe how such a colorimetric test might work,specifically addressing how you would detect a sample that has beentainted with chondroitin sulfate.

Explanation / Answer

Chondroitin sulfate is a ubiquitous component ofproteoglycans that is present both in the extracellular matrix andat the cell surface of various tissues. Until recently, chondroitinsulfate has attracted less attention than heparan sulfate anddermatan sulfate, owing to the limited number of known chondroitinsulfate-binding proteins. To determine the biological function ofchondroitin sulfate, biotinylated probes were prepared and used tosearch for binding proteins. Chondroitin sulfates A, C, D, and E were biotinylated througheither the uronic acid or the residual core peptide. Lysates frommouse Lewis lung carcinoma (3LL) cells were blotted onto anitrocellulose membrane after sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and the membrane was treated withthe biotinylated chondroitin sulfates. Among the chondroitin sulfate variants, the E type showed the mostintense bands upon visualization of the membrane withavidin-conjugated alkaline phosphatase and the appropriatesubstrates. The binding of chondroitin sulfate E to proteins in thecell lysate was not affected by the A, C or D variants but wasreduced by treatment with dermatan sulfate. Lysates from 3LL cellswere also treated with biotinylated chondroitin sulfate E and,after two-dimensional (2-D) electrophoresis and blotting, severalchondroitin sulfate E-binding proteins including lamins andheterogeneous nuclear ribonucleoproteins were identified by massspectrometry.

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