mast cell heparin isolation

Dr. Tilo Brunnee tilo at brunnee.IN-Berlin.DE
Wed Oct 18 15:27:09 EST 1995


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!

---------
Dr. Tilo Brunnee, physician, Allergist
Dept. of Clinical Immunology and Asthma Outpatient Department
Humboldt-University Berlin, Germany       e-mail: tilo at brunnee.in-berlin.de




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