Contration of primary amine by TNBS assay

TNBS assay

TNBSA Solution ( 5% solution of trinitrobenzene sulfonic acid in methanol) TNBS reacts with primary amines (peptides or amino acids) to yield a soluble colored product, a property useful for various assay methods. TNBSA component reacts readily with primary amino groups of amino acids in aqueous format at pH 8 to form yellow adducts Colored derivatives are monitored at 345nm and have extinction coefficients of 10,000 to 15,000

2,4,6-Trinitrobenzene sulfonic acid 5% w:v in MeOH essentially insoluble in water; soluble in methanol

TNBS reacts with primary amines molecules to from a highly chromogenic (orange) product, whose absorbance at 335 to 345nm can be measured with a plate reader or spectrophotometer. TNBSA can be used for measuring amines, sulfhydryls, hydrazides, and ε-amino groups of Llysine.

1) Dilute supplied 5% TNBSA solution 500-fold in 0.1M sodium bicarbonate buffer (pH 8.5). rem : do not prepare this working solution in advance. Use immediately. 2) Add 250µL diluted TNBSA solution to 500µL of protein solution. Mix well. rem: sample should be at 20-200µg/ml (proteins) or 2-20µg/ml (aminoacids) in a compatible buffer (devoid of Tris or other amine). Proteins car be dissolved if lyophilized or dialyzed in 0.1M sodium bicarbonate buffer (pH 8.5). 3) Incubate at 37°C for 2 hours. 4) Add 250µL of 10% SDS and 125µL of 1N HCl to each sample to stop and stabilize reaction. 5) Measure absorbance of solution at 335nm. Determine concentration of primary amines by calculation from the extinction coefficient or by comparison to amino acid standards.

Standard amine solutions with a known amount of reagent and the test solutions containing unknown amounts of the primary amine derivative were serially diluted in duplicates with 0.1 M sodium tetraborate to give a final volume of 100 µl in a 96 well plate. Concentrations of approximately 17.5 to 210 nmol/ml amine reagent were achieved and then 2.5 µl of TNBS (75 nmol) in water were added to each well. After 5-20 minutes incubation time at room temperature (depending on the strength of the developed colour) the absorption was measured at 405 nm using a microplate reader. 

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 4oC in brown bottle. Make up to 1.1M fresh daily (100mg recrystallized TNBS in 0.2ml H2O).

METHOD: make fresh daily Solution D: 1.5ml Solution A + 98.5ml Solution B standard curve: BSA at 0.1, 0.2, 0.5, 1.0, and 2.0 mg/ml in 0.25ml H2O. samples: same concentration range as above in same volume. add 0.25ml solution C add 10µl TNBS (take note of time!) Incubate exactly 5 min at 23oC. add 1ml solution D to stop reaction. 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.