The pH dependence of the binding of pro-urokinase to fibrin/celite.
A re-examination of the affinity of pro-urokinase (pro-UK), HMW and LMW-urokinase (UK) to fibrin/Celite was undertaken in order to explain how the chance purification of pro-UK from freshly voided urine by fibrin/Celite affinity chromatography may be reconciled with the subsequent observations that pro-UK failed to bind significantly to fibrin clots in plasma. A significant pH dependence of pro-UK binding to fibrin/Celite was found. Substantial binding of pro-UK (native or recombinant from E. coli), but not of the two-chains forms, was seen at about pH 6.5, which is in the normal pH range of pooled, freshly voided urine. By contrast, at pH 7.4 fibrin binding of pro-UK was much reduced, though it was still significantly greater than that of HMW or LMW-UK. This finding helps to explain the fibrin-binding of pro-UK in freshly voided urine but not in blood. In order to determine if this pH dependence was the sole explanation for why pro-UK could not be isolated by this method from stored urine, the stability of pro-UK in urine was evaluated by incubating 125I-labeled pro-UK in urine. Incubation for up to 4 days (37 degrees C) was not accompanied by any degradation of the single-chain pro-UK as evidenced by autoradiography under reducing conditions. It was concluded that the alkaline shift in pH which occurs in urine left standing, rather than the degradation of pro-UK, explained why freshly voided urine was found to be essential. Clot lysis studies at pH 6.5 and 7.4 showed no promotion of fibrinolysis at the pH which favored fibrin/Celite binding. Therefore, while the present study defines the conditions under which pro-UK may be purified from urine by fibrin/Celite chromatography, it provides no evidence that this binding phenomenon plays any role in fibrinolysis.
Pannell
R
R
Vascular Research Laboratory, New England Deaconess Hospital, Harvard Medical School, Boston, MA 02215.
Angles-Cano
E
E
Gurewich
V
V
D007692:Diatomaceous Earth / Q000378:metabolism*; D004795:Enzyme Stability; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010960:Plasminogen Activators / Q000378:metabolism* / Q000652:urine; D011485:Protein Binding; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism* / Q000652:urine
D007692:Diatomaceous Earth; D005337:Fibrin; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator