Lidstrom:Solution Stock Info

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ATP

Recommendations

• ATP, sodium salt (MW 551)
  • Store desiccated powder @ -20°C
  • In H2O, bring to pH 7, with 2 equiv NaOH, store -20°C
  • Stable for months with minimal concentration loss

NAD(P)/H

• NAD(P)H are rapidly destroyed in acid in conditions where NAD(P)+ are completely intact
• 99% destruction of NADH
  • @23°C = 1.2 min @ pH 2, = 2 hr @ pH 4
  • @38°C, 3-4x faster
  • @60°C, 20x faster
• NADPH is destroyed faster than NADH (~80% faster @ 30°C)

• @pH 2, NAD+ is 100,00x more stable than NADH
  • Allows elimination of reduced form by pH drop
• Above pH 7, degradation rate of NAD increases,5000x faster at pH 12.5-13
• NADP+ appears to be more stable than NAD+
• Higher temp, more salts increase degradation rate

• NADH becomes more stable with pH increases, excluding low concentration in small volumes
• Oxidation is a problem above pH 8
• @ pH 7 25°C, rate of destruction in ~0.2%/hr, increases 10x @ pH 6

• Strong solutions (40 mM) of NADH store well @ pH 9-12, but large losses @ 4°C
• Weak solutions (0.4 mM) of NADH store well @ 4°C from pH 9-11, destroyed @ -20°C pH >10.5
• Similar characteristics for NADPH

• NADH presence in small volumes is sensitive to oxidation at neutral pH
  • increases ~inverse square root of the volume
  • can prevent with 1 to 2 mM ascorbic acid

Recommendations

• Store pyridine nucleotides, desiccated @ -20°C
• Can store NAD+/NADP+ solution for months without significant concentration change/
• Can standardize stock solutions with spectrophotometer
• Dissolve NAD+/NADP+ in water, ~100 mM
• Unless < -40°C, NAD(P)H should be prepared at < 5 mM, pH 9-11 and stored at 4°C. At low temp, strong solution can probably be stored without too much loss

Citations

Passonneau, J. V., Lowry, O. H., & Lowry, O. H. (1993). Enzymatic analysis: A practical guide. Totowa, NJ: Humana Press.