amide gel electrophoresis (1). We have shown (2) by
We have established a fluorescence method to detect the overlay technique that calcium ions bind to concalcium-binding proteins by making use of the quinonectin (also called titin), which is a giant myofibrillar line Ca indicator quin2. Authentic calcium-binding protein with molecular mass of about 3000 kDa . proteins were subjected to sodium dodecyl sulfate-This assay, however, needed over 1 week for sufficient polyacrylamide gel electrophoresis and then electroexposure to X-ray film. Moreover, it requires handling phoretically transferred onto polyvinylidene difluorthe radioisotope, accompanied with a troublesome danide membranes. Transfers were incubated with nonrager of exposure to radiation. dioactive calcium ions, then with quin2 to detect the We were able to overcome these problems by making calcium-binding proteins as fluorescent bands by illuuse of the fluorescent quinoline Ca indicator quin2, mination with UV light. Calmodulin and parvalbumin which was rationally designed and chemically syntheof EF hand conformation calcium-binding type were sized by Tsien (5). Quin2 (8-amino-2-[(2-amino-5-methclearly identified. Quin2 distinguished smooth muscle ylphenoxy)methyl] -6 -methoxyquinoline -N, N, N, Na-actinin from skeletal muscle a-actinin; the former tetraacetic acid, tetrapotassium salt) was originally was faintly fluorescent, having a low affinity for calused as a fluorescent signal for quantitative determinacium ions. In whole myofibril preparations from skeletion of intracellular free calcium ion concentrations tal muscles, troponin-C, connectin (titin), and nebulin with good time resolution. It has an extremely high were intensely fluorescent, being shown to have calselectivity for calcium ions; quin2 binds to calcium ions cium-binding ability. The fluorescence method is an with 1:1 stoichiometry at the binding constant of 1.3 1 accurate, safe, and simple procedure to detect the 10 7 . Its affinity for magnesium ions is negligibly low