Influence of the high affinity divalent cation on actin tryptophan fluorescence.
This study demonstrates that the intrinsic tryptophan fluorescence of actin provides an effective and convenient way of measuring divalent cation exchange and polymerization of native actin. In the measurement of divalent cation exchange, this method is as sensitive as those previously used (8) and provides further evidence of the importance of the tightly bound divalent cation to the properties of actin. In monitoring polymerization, this method cannot compete with the sensitivity of the commonly used pyrene-actin fluorescence. However, some proteins (e.g., profilin) and other agents (e.g., cytochalasin D) that bind to actin are affected by the presence of fluorescent labels, making actin intrinsic fluorescence potentially useful in investigating the interaction of these agents with actin, and in validating data obtained using labeled actin. Our laboratory routinely checks the quality of our native actin preparations using this technique and, more recently, we have used actin tryptophan fluorescence to monitor the nucleating and severing effects of gelsolin on actin. The simplicity of the technique is most appealing, and we expect that a variety of innovative and routine uses will be developed.
Selden
L A
LA
Research Service, Stratton VA Medical Center, Albany, New York 12208.
Kinosian
H J
HJ
Estes
J E
JE
Gershman
L C
LC
D000199:Actins / Q000737:chemistry*; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000494:pharmacology*; D002413:Cations, Divalent; D005453:Fluorescence; D005456:Fluorescent Dyes; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000494:pharmacology*; D009282:Naphthalenesulfonates; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000199:Actins; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D014364:Tryptophan; D008274:Magnesium; D002118:Calcium