Intramolecular distance measurements in alpha-lactalbumin.
The distance between the calcium site (site I) and the zinc site (site II) in alpha-lactalbumin was estimated from Forster energy-transfer measurements between donor Eu(III) [or Tb(III)] at site I and acceptor Co(II) at site II to be 11.5 +/- 1.5 A. Intersite distances were also measured between the bis-ANS [4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]] binding locus and cobalt at site II (13.6 +/- 1.0 A), between bis-ANS and a fluorescein moiety covalently bound to Met-90 (33.5 +/- 3.0 A), and between Met-90 (fluorescein) and cobalt at site II (16.7 +/- 1.0 A). The apparent Kd for cobalt binding to site II agreed well with the value measured previously by intrinsic fluorescence [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. A Zn(II) titration of Eu(III)-alpha-lactalbumin reconfirmed that both sites I and II can be occupied simultaneously [Musci, G., & Berliner, L. J. (1985) Biochemistry 24, 3852-3856], since the lanthanide fluorescence was unaffected.
D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D004735:Energy Transfer; D019793:Fluorescein; D005452:Fluoresceins; D007768:Lactalbumin / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence / Q000379:methods; D015032:Zinc / Q000378:metabolism
D000817:Anilino Naphthalenesulfonates; D005452:Fluoresceins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D007768:Lactalbumin; D015032:Zinc; D002118:Calcium; D019793:Fluorescein