Tryptophan fluorescence as a probe of placental ribonuclease inhibitor binding to angiogenin.
The binding of human placental ribonuclease inhibitor (PRI) to angiogenin, a human protein that induces neovascularization, occurs with a 1:1 stoichiometry and is accompanied by a 50% increase in tryptophan fluorescence. In contrast, the binding of PRI to bovine pancreatic RNase A or to angiogenin oxidized at its single tryptophan residue results in a quenching of fluorescence. These observations suggest that there is a change in the local environment of Trp-89 of angiogenin. Quenching experiments with acrylamide are consistent with the view that Trp-89 is exposed in the native protein and becomes less accessible upon formation of the complex with PRI. Stopped-flow kinetic measurements monitoring the fluorescence enhancement indicate a two-step mechanism for the binding of PRI to angiogenin. The first step involves rapid formation of an enzyme-inhibitor complex, EI, followed by a slower isomerization of EI to a tight enzyme-inhibitor complex, EI*: (Formula: see text). In 0.1 M NaCl at pH 6 and 25 degrees C, the values of K1 and K2 are 0.53 microM and 97 s-1, respectively. The apparent second-order rate constant of association at protein concentrations much less than K1 is approximated by K2/K1 and equals 1.8 X 10(8) M-1 s-1. The corresponding value for the association of PRI with RNase A is only slightly higher, 3.4 X 10(8) M-1 s-1. The effects of pH and sodium chloride concentration on the association rate of PRI with angiogenin suggest the importance of ionizable groups and ionic interactions, respectively, in the association process.(ABSTRACT TRUNCATED AT 250 WORDS)
Lee
F S
FS
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115.
Auld
D S
DS
Vallee
B L
BL
D043925:Angiogenesis Inducing Agents / Q000378:metabolism*; D001699:Biometry; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010926:Placental Hormones / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000037:antagonists & inhibitors*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D043925:Angiogenesis Inducing Agents; D006133:Growth Substances; D010926:Placental Hormones; D011506:Proteins; C022052:placental ribonuclease inhibitor; D014364:Tryptophan; D012260:Ribonucleases; C046366:angiogenin; D012259:Ribonuclease, Pancreatic