Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Fluorescence Methods for Ca 2+ Exchange 101
chelators that undergo changes in absorption (or fluorescence) upon Ca 2+ -binding
(as discussed by S. Linse in Chapter 2).
13. The rates of Ca 2+ dissociation from proteins can also be determined using the
luminescent trivalent cation terbium (Tb 3+ ). Tb 3+ undergoes a large increase in
fluorescence (an phosphorescence) upon binding to Ca 2+ -binding proteins. When
Tb 3+ is reacted with proteins containing bound Ca 2+ (or Mg 2+ ), its luminescence
increases at the rate of Ca 2+ (or Mg 2+ ) dissociation. We have used Tb 3+ fluorescence
and stopped-flow methodology to determine the rates of Ca 2+ and Mg 2+
dissociation from parvalbumin and EGTA (16). Ca 2+ exchange rates can also be
determined by NMR spectroscopy (see T. Drakenberg, Chapter 18).
14. We have recently introduced a method that allows the generation of Ca 2+ transients
of various amplitudes and duration in a stopped-flow apparatus. This
method is based on the fact that EGTA and Mg-EDTA have a slow Ca 2+ on-rate
and when Ca 2+ is rapidly mixed with a solution containing EGTA (or Mg-EDTA),
[Ca 2+ ] transiently rises until it is bound by chelator. This has allowed us to produce
“artificial” Ca 2+ transients which vary in duration from 0.1 to 50 ms and to
observe the transient activation of various Ca 2+ -binding proteins and Ca 2+ dependent
enzymes in response to these transients (15). Thus, using stopped-flow techniques
similar to those discussed above it is possible to produce Ca 2+ transients
and follow the response of Ca 2+ -binding proteins to these transients in a stoppedflow
apparatus.
Acknowledgments
This work was supported by a grant from the National Institutes of Health
(DK33727).
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