Protein Analysis
High-Yield Summary
- Protein analysis covers 4 goals: structure, amino acid composition/sequence, functional activity, and concentration.
- Structure: X-ray crystallography (crystallize + diffraction pattern, high resolution) vs. NMR spectroscopy (magnetic nuclei, in solution, smaller proteins).
- Simple hydrolysis reveals composition only (which amino acids, how much); Edman degradation reveals sequence — removes/identifies one residue at a time from the N-terminus.
- Edman degradation has a practical accuracy limit (~30 residues) well below its outer bound (~50–60) — incomplete cleavage compounds error each cycle.
- Enzyme activity is measured by tracking a color-change reaction the enzyme catalyzes over time.
- Concentration: UV spectroscopy (280 nm, Trp/Tyr absorbance — fast but contaminant-sensitive) vs. colorimetric assays (BCA, Lowry, Bradford — color change proportional to protein amount, used to build a standard curve).
Key Terms
- X-ray crystallography
- Crystallize protein, analyze X-ray diffraction pattern for high-resolution 3D structure.
- NMR spectroscopy
- Uses magnetic properties of atomic nuclei to reveal structure of smaller proteins in solution.
- Edman degradation
- Sequential removal/identification of one amino acid at a time from a peptide's N-terminus.
- Bradford assay
- Colorimetric protein assay; Brilliant Blue dye shifts brown→blue on binding protein, proportional to concentration.
- Standard curve
- Calibration curve (known concentrations vs. color/absorbance) used to find an unknown sample's concentration.
X-ray Crystallography vs. NMR Spectroscopy
| X-ray Crystallography | NMR Spectroscopy |
|---|---|
| Requires crystallized protein | Protein studied in solution |
| High-resolution detail | Best for smaller proteins |
Simple Hydrolysis vs. Edman Degradation
| Simple Hydrolysis | Edman Degradation |
|---|---|
| Reveals composition (which amino acids, how much) | Reveals exact sequence, N-terminus first |
| No length limit concern | Practical accuracy limit ~30 residues (outer bound ~50–60) |
Determining Protein Concentration by Colorimetric Assay
- 1Prepare known-concentration protein standards and the unknown sample.
- 2Add the colorimetric reagent (e.g., Bradford dye) to each.
- 3Measure the resulting color intensity/absorbance for each standard.
- 4Plot standards to build a standard curve (concentration vs. color).
- 5Read the unknown sample's color intensity against the standard curve to determine its concentration.
Common MCAT Trap
- Edman degradation's theoretical max (~50–60 residues) is NOT the same as its reliable practical limit (~30) — a passage citing a large number may be testing whether you know accuracy degrades well before the outer bound.
- UV spectroscopy absorbance at 280 nm comes from aromatic residues (mainly Trp and Tyr, Phe only minimally) — not from the peptide bond itself.
- Simple hydrolysis and Edman degradation answer different questions — composition vs. sequence — don't treat them as interchangeable.
Quick Recall
Which structural determination method works with the protein in solution, and which requires a crystal?
Which two amino acids are primarily responsible for UV absorbance at 280 nm?
What causes Edman degradation's accuracy to degrade over a long peptide?
Name the three common colorimetric protein assays.