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mast cell heparin isolation

Steven Pirie-Shepherd srps at galactose.mc.duke.edu
Thu Oct 19 06:48:45 EST 1995

Dr. Tilo Brunnee (tilo at brunnee.IN-Berlin.DE) wrote:
: Hallo!
: I attempt to isolate human mast cell heparin proteoglycan from human lungs.
: So far I enzymatically and mechanically prepare a single cell suspension
: from human lung, add 4 M Guanidine HCl and sonicate to disrupt the cells
: and dialyze against 1M NaCl/10mM Phosphate, pH 6.0 and apply this soup on a
: Dowex 1x2-200 ion exchange column equilibrated with the same buffer, elute
: with 3M NaCl/10mM Phosphate, dialyze against H2O and speedvac to dryness.
: I do have some questions regarding heparin:
: - Are there more efficient/more gentle ways to prepare highly sulfated
: proteoglycans?
: - Are ther any contaminants (that bind to strong ion exchange at pH 6 in
: the presence of 1 M NaCl?) to expect from crude human lung?
: - Is it true that heparin side chains are bound to a protein core in human
: mast cells?
: - If so, is the heprain sidechain cleaved from the protein core during or
: before degranulation?
: - Is there any knowledge about the average lengh of the heparin molecules
: (or molecular weight) in humans?
: - Is there another source of heparin than mast cells in humans?
: - I wonder how commercially available heparin is isolated. That material
: seem to consist only of sulfated carbohydrates of relatively homogeneous
: size.

: I would be very grateful for some enlightment. Thank you!

If I wre you I would find a lot of references by Prof HRP Miller 
(University of Edinburgh). His group deals a lot with Heparin, Mast 
cells, and the associated proteins. There is also a prof Allen at 
Newcastle who has techniques from dealing with GAGS, proeoglycans etc. 
Medline searches should get you tthe appropriate references.


Steven Pirie-Shepherd		       		
srps at galactose.mc.duke.edu	        
"Insert your own pithy phrase just about here!"

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