Relationship between annexin V tryptophan exposure, calcium, and phospholipid binding.
Annexin V is a Ca(2+)-dependent phospholipid-binding protein that may have one or more membrane-related functions, including inhibition of blood coagulation. The fluorescence of the single tryptophan of annexin V was used to monitor Ca2+ and/or phospholipid binding in terms of emission wavelength, emission intensity, and susceptibility to acrylamide quenching. In the absence of phospholipid, Ca2+ titration showed a strong red shift of the wavelength of maximal emission to approximately 345 nm, where a small increase in intensity occurred and was half maximal at approximately 3 mM Ca2+. The Stern-Volmer quenching constant due to acrylamide was only 5.2 M-1 for annexin V alone, indicating limited aqueous exposure of the tryptophan, but 36 M-1 for a Ca(2+)-bound form, indicating full exposure. Binding to both negatively charged and zwitterionic phospholipids was accompanied by a very large increase in fluorescence emission intensity, a red shift, and low exposure to acrylamide. Calculated concentrations of Ca2+ near the surface of negatively charged vesicles suggested that the exposure of tryptophan by Ca2+ binding to annexin V was sufficient for binding of the protein to all vesicles tested, including those composed of oleic acid and phosphatidylcholine (PC), but not to those composed of pure PC. When binding to PC was monitored, the phenomena associated with phospholipid binding were observed separately, at higher Ca2+ concentration, from the red shift and the high exposure to acrylamide due to Ca2+ binding alone.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pathology, Boston University School of Medicine, Massachusetts 02118.
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D017304:Annexin A5 / Q000737:chemistry* / Q000378:metabolism; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008823:Micelles; D019301:Oleic Acid; D009829:Oleic Acids; D010713:Phosphatidylcholines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence; D013096:Spermine / Q000494:pharmacology; D014364:Tryptophan / Q000737:chemistry*
D017304:Annexin A5; D008081:Liposomes; D008823:Micelles; D009829:Oleic Acids; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; D013096:Spermine; D019301:Oleic Acid; D014364:Tryptophan; D002118:Calcium