Estimation of Amino Groups Using TNBS

Reference:

Fields, R. 1972. Methods in Enzymology. 25:464-469.

Materials:

Solution A: 100mls of 0.1M Na₂SO₃ (fresh each week)Solution B: 1.0l of 0.1M NaH₂PO₄

Solution C: 1.0l of 0.1M Na₂B₄O₇ in 0.1M NaOH (make up in acid and ddH₂O- washed glass).

Trinitrobenzene sulfonate (TNBS) 10g in 10ml H₂O, heat to dissolve and remove black flecks of oil by centrifugation. Add HCl to 2M and cool to room temp. Wash the crystalline precipitate on a glass filter with 1M HCl. Desiccate and store at 4^oC in brown bottle. Make up to 1.1M fresh daily (100mg recrystallized TNBS in 0.2ml H₂O).

Method:

1. Make fresh daily Solution D: 1.5ml Solution A + 98.5ml Solution B
2. Standard curve: BSA at 0.1, 0.2, 0.5, 1.0, and 2.0 mg/ml in 0.25ml H₂O.
3. Samples: same concentration range as above in same volume.
4. Add 0.25ml solution C
5. Add 10µl TNBS (take note of time!)
6. Incubate exactly 5 min at 23^oC.
7. Add 1ml solution D to stop reaction.
8. Measure OD₄₂₀. Standard curve should range from about 0.09 to >1.8

Calculation:

OD₄₂₀ = 1.0 = 52nmol amino groups = 78nmol/1.5ml assay mix

“1.0mg/ml” BSA sample (0.25mg) = OD₄₂₀ of 0.945 = 73.41nmol NH₃

therefore, 0.25 mmol BSA = 73.41 nmol NH₃ groups

67,000

3.73nmol BSA = 73.41 nmol NH₃

therefore 1 molecule BSA = 19.8 molecules of lysine.