Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Enzymatic Assays 347
5. Add 20 nM NOS (see Notes 5 and 6), mix and continue to record A 401. The
absorption may increase slightly due to the low basal activity of NOS, as shown
in Fig. 3 (see also Note 5).
6. Add the desired [CaM], mix, and continue to record A 401. Upon addition of CaM,
NOS will be activated and, as it produces NO, A 401 will increase. Figure 3 shows
an approx 10-fold enhancement in the rate of increase of A 401 upon the addition
of 50 nM CaM.
7. Inhibitors of CaM or NOS can be added during the linear phase of the increase in
absorption and inhibition can be followed by their ability to prevent further
increases in A 401 (see Note 2). This is demonstrated in Fig. 3, where 1 µM of a
CaM isoform (SCaM-1), which is a competitive antagonist of NOS (3), was added
to inhibit NOS.
8. NOS’s enzymatic activity can be calculated from the rate of change in A 401 using
∆ε = 38 mM/cm.
3.4. NOS Oxidation of NADPH
NADPH undergoes a decrease in absorption at 340 nm (A 340) upon oxidation
and this provides a convenient method for following this function of NOS’s
reductase domain. In the absence of exogenous electron acceptors (cyt c or
FeCN), the rate of NADPH oxidation is also dependent upon the presence of a
functional electron acceptor (heme) in NOS’s oxidase domain (see Note 7).
To conduct these assays:
1. Make up 11 mL of 50 mM HEPES (pH 7.5), 3 mM DTT, 1 mM L-Arg, 1 mM
CaCl 2, 4 µM FAD, FMN, H 4B, and 110 U catalase.
2. Pour the prepared solution into a preweighed 1 mg vial of NADPH. Mix by inverting
five times and preincubate at the desired reaction temperature (see Note 4).
3. Pipet approx 1 mL (less the volume of NOS to be added later) of the above mixture
into a cuvet, and follow A 340 for 2 min in a UV/VIS spectrophotometer. This
may show a slight linear decrease if there is basal activity.
4. Add 20 nM NOS, mix rapidly and continue to record A 340. This absorption may
decrease slightly because of the basal activity of NOS.
5. Add the desired [CaM], mix rapidly and continue to record A 340. Upon addition
of CaM, NOS will be activated and its rate of NADPH oxidation should increase
resulting in a faster decrease in A 340. Depending on the purity of the NOS, you
can expect a 5–100-fold increase in the rate of NADPH oxidation upon addition
of CaM.
6. Inhibitors of CaM or NOS can be added during the linear phase of the increase in
A 340 and their inhibition can be followed by their ability to prevent further
decreases in absorbance (see Note 2).
7. Enzyme activation can be calculated from the rate of decrease in A 340 using ∆ε =
6.22 mM/cm.
8. It is also possible to conduct the NADPH oxidation assay in the presence of an
exogenous electron acceptor like cyt c (see Note 7).