Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

348 Walsh et al.
3.5. NOS Reduction of Cyt c
Activated NOS can transfer electrons from its reductase domain directly to
exogenous electron acceptors like cyt c. The increase in cyt c absorbance at
550 nm provides a continuous assay of this reductase domain function of NOS.
To conduct this assay:
1. Make up 11 mL of 50 mM HEPES (pH 7.5) and 1 mM CaCl 2 and let it equilibrate
to the desired temperature.
2. Pipet approx 1 mL of this solution (less the volume of cyt c and NOS to be added
later) into a cuvet and add cyt c (50 µM) and NOS (10–20 nM).
3. Mix, then place the cuvet in the spectrophotometer, and follow A 550 for 2 min.
This baseline should be a stable flat line.
4. Add NADPH (100 µM), mix rapidly, and continue to follow A 550. Upon addition
of NADPH, the absorption should show a linear increase (see Note 8).
5. Quickly add the desired [CaM]. Rapidly mix and continue to follow A 550. After the
CaM addition the rate of increase in A 550 should accelerate. Under the conditions
described, the reaction will remain linear for only 4–5 min. Depending on the purity
of the NOS you can expect a 5–25-fold increase in rate compared to basal.
6. Enzymatic activity can be calculated from the rate of increase in A 550 using ∆ε =
21 mM/cm.
3.6. NO Production as Measured by the Citrulline Assay
This assay is based on the quantitative determination of radioactive citrulline
formed by NOS from L-[ 14 C]Arg. Although the L-Arg substrate binds to
the cation exchange resin (because of its positively charged guanidino moiety),
radiolabeled L-citrulline will flow through.
To conduct this assay:
1. Make up 11 mL of 50 mM HEPES (pH 7.5) and let it equilibrate to the desired
temperature.
2. Add 200 µM L-[ 14 C]Arg, 1 mM CaCl 2 , 4 µM FAD, FMN, H 4B.
3. Pour the prepared solution into a preweighed 1 mg vial of NADPH and mix by
inverting five times.
4. Pipet approx 300 µL (less the volume of NOS and CaM to be added later) of the
aforementioned mixture into plastic tubes.
5. Add the desired [CaM] to each tube. Vortex and preincubate at 37°C for 5 min.
6. Start the reaction by adding 10–25 nM NOS. Vortex each tube quickly and incubate
at 37°C for 5–20 min, depending on the time over which the reaction is
linear (see Note 2).
7. After incubation for a precise time, stop the reaction by adding 5 µL of 6 N trichloroacetic
acid to each tube. Vortex and place on ice.
8. Neutralize the pH by adding 250 µL of 1.5 M HEPES (pH 7.5) to each tube.
9. Apply the contents of each tube to a separate 1 mL cation exchange column
(sodium form).