Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Enzymatic Assays 341
by CaM, we describe four assays that measure these different CaM-dependent
catalytic activities: an oxyhemoglobin (HbO 2) assay and a citrulline assay that
measure NO synthesis occurring in the heme domain (12,13); an NADPH oxidation
assay that measures this reductase domain function that is also dependent
on a functional heme domain (12,13); and a cyt c reduction assay that
selectively measures the function of NOS’s reductase domain (14).
2. Materials
2.1. Phosphodiesterase Assay
1. A spectrofluorimeter that can monitor fluorescence intensity as a function of time.
2. Quartz cuvets.
3. Purified CaM or CaM mutant (100–300 µM stocks that are stable for years if
frozen).
4. The fluorescent substrate, 2'-methylanthraniloyl-cyclic GMP (Mant-c-GMP).
Stable for years if frozen as a 2.5-mM stock.
5. 10 mM MOPS, 200 µM EGTA, 90 mM KCl, 0.5 mM CaCl2, 5 mM MgCl2 (pH 7.0).
6. Purified PDE (approx 1 mg/mL). Stable for years at –20°C in 50% glycerol.
2.2. Calcineurin Assay
1. The spectrofluorimeter, cuvets, and CaM stocks aforementioned.
2. The fluorescent substrate, 4-methylumbelliferyl phosphate (MUF). Stable for
years if frozen as a 10-mM stock in the absence of contaminating phosphatases.
3. 50 mM Tris-HCl, 200 µM EGTA, 0.5 mM CaCl 2, 5 mM MgCl 2 (pH 7.4). This
buffer should be boiled for 10 min to denature contaminating phosphatase.
4. Purified CaN (0.5–1 mg/mL). Stable for years if frozen in aliquots to avoid freezethawing.
2.3. NOS Oxyhemoglobin Assay
1. UV/VIS spectrophotometer capable of recording absorption as a function of time.
A temperature control device is optional. Disposable 1-mL plastic cuvets can be used.
2. 50 mM HEPES buffer (pH 7.5).
3. HbO 2 (approx 300 µM stock solution), prepared as a 12.5 mg/mL solution of
commercially available >95% pure HbO 2 (Sigma) in 50 mM HEPES (pH 7.5)
and stored at –80°C.
4. Dithiothreitol (DTT) (0.3 M stock in double-distilled water [ddH 2O]).
5. L-Arg (1 M stock in ddH 2O).
6. CaCl 2 (0.5 M stock in ddH 2O).
7. NADPH (preweighed 1-mg vials). Stable for months if stored dry at room temperature.
8. Flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and
tetrahydrobiopterin (H 4B) (approx 4 mM stock in ddH 2O).
9. Catalase (100,000 U/mL stock in ddH 2O).
10. Superoxide dismutase (SOD; 10,000 U/mL stock in ddH 2O).