Biochemistry
Biochemistry tests the structure and function of biological molecules, acid-base behavior, bioenergetics, and quantitative assays.
Core rules
- Carbohydrates, lipids, proteins, and nucleic acids are built by condensation and broken by hydrolysis. Reducing sugars have a free carbonyl group and reduce copper or silver reagents.
- Proteins have primary to quaternary structure; denaturation disrupts noncovalent bonds and usually destroys activity without breaking peptide bonds.
- DNA contains deoxyribose and thymine; RNA contains ribose and uracil. Base pairing and stacking stabilize duplex DNA.
- Buffers resist pH change near their pKa; the Henderson-Hasselbalch equation relates pH, pKa, and the ratio of conjugate base to acid.
- Metabolism couples exergonic reactions to endergonic ones, often through ATP or NADPH.
- Spectrophotometry uses Beer-Lambert law, A = epsilon c l, but absorbance is not a direct concentration without a standard or known extinction coefficient.
Worked example
A protein solution gives A280 = 0.45 in a 1 cm cuvette. The extinction coefficient is 15,000 M^-1 cm^-1. Rearranging A = epsilon c l gives c = 0.45 / (15,000 x 1) = 3.0 x 10^-5 M, or 30 micromolar. If the sample were turbid, the reading would overestimate protein, so clarification and a blank are needed.
Traps
Do not confuse absorbance with transmittance. Do not assume A260/A280 is pure DNA without checking contamination. Do not say denaturation breaks peptide bonds. Do not ignore the buffer's temperature and concentration dependence. Do not use a reducing-sugar test to quantify all carbohydrates.