SI-BF-100
World Precision Instruments
23
calcium concentrations. This is because less calcium is bound to the dye, and
calcium-free dye is excited more effectively by longer wavelengths of light. The ratio
between the fl uorescence intensities caused by 340nm and 380nm of light is an
accurate indicator of the concentration of calcium in the fi bers.
Fig. 41— The fl uorescence intensity of Fura-2 as a function of calcium concentration
when excited by different wavelengths of light. The colored bars indicate the location of
fl uorescence intensities caused by excitation with 340nm and 380nm light. Fluorescence
was recorded at 510nm.
APPENDIX B: EXAMPLE OF ATPASE ACTIVITY
MEASUREMENT
Muscle contraction and relaxation is caused by the attachment and detachment
of two types of molecules ( actin and myosin) to each other within the fi bers that
compose a muscle. Each crossbridge that is made between the end of a myosin
molecule and a binding site on an actin molecule requires a molecule of ATP, an
energy source, to be hydrolyzed when the end of the myosin molecule is released
from the actin fi lament. After its release, the myosin molecule is ready to move over
to another actin binding site causing the sarcomere, and the muscle, to shorten.
When ATP is regenerated through a series of enzymatic reactions, the molecule,
NADH, provides the energy needed for this regeneration. The reduced form of
NADH fl uoresces when exposed to ultraviolet (UV) light. However, when NADH is
oxidized, and its stored energy is released for the regeneration of ATP, the oxidized
form of the molecule, known as NAD, does not fl uoresce. Therefore, any reduction
in the fl uorescence of the NADH-containing solution that is surrounding the muscle
preparation indicates an increase in the activity of the enzyme (ATPase) that releases
the energy of the ATP molecule for use in muscle contraction.
This technique can only be performed on skinned muscle fi bers, which are fi bers
