Trinitrobenzenesulfonic acid hydrate (TNBS) reacts with amino acids, yielding a yellow product (Fig. B2.2.3) whose absorbance is measured at 340 nm. TNBS reacts only with amino groups in their unprotonated state. TNBS binds to amino acids in two steps: (1) a fast reaction with low affinity, and (2) a slow reaction with relatively high affinity. For practical purposes, the two-step reaction should be considered as one. TNBS reacts with primary amines under slightly alkaline conditions, and lowering the pH stops the reaction.
Additional Materials (also see Basic Protocol 1) 1% (v/v) SDS (APPENDIX 2A)
0.2 M sodium phosphate buffer, pH 8.2 (appendix 2a) 2.5 mM leucine standard in 1% SDS
0.1% (w/v) trinitrobenzenesulfonic acid dihydrate (TNBS) in deionized water
(prepare immediately before use and protect from light) 0.1 N HCl
15 x 85-mm and 16 x 125-mm glass test tubes 50° and 75°C shaking water baths Spectrophotometer (visible range; 340 nm)
1. Determine the appropriate amounts of substrate and enzyme to use for the hydrolysis reaction. Use an amount of substrate that gives protein at 8% (w/w) of the total reaction and calculate enzyme at a ratio of 0.012 Anson U/g protein. For example, for a 100-ml Alcalase hydrolysis reaction, use:
substrate containing 8 g protein
0.16 g Alcalase 0.6L or 0.04 g Alcalase 2.4L
buffer to 100 ml.
The amount of protein in the substrate solution can be determined by using the Kjeldahl method (unit bi.2) to determine the nitrogen concentration (N) and then multiplying this by a factor of 6.25 (fN).
The mass of enzyme in frams (ME) is caluculated ME = MP x (A/E), where (MP) is the mass (in g) of protein in the substrate, A is the suggested enzyme activity in Anson units/gram (0.012 AU/g), and E is the actual enzyme activity specified by the manufacturer. For Alcalase, 0.6L and 2.4L correspond to 0.6 and 2.4 AU/g, respectively. Thus ME = 8 g x (0.012 AU/g 0.6 AU/g) = 0.16 g for 0.6L, or 0.04 g for 2.4L.
2. Mix substrate and appropriate buffer at the desired temperature and pH.
Every sample should be analyzed with appropriate replicates.
3. Before adding enzyme, take a 0.25-ml sample and transfer to a 15 x 85-mm glass test tube containing 2 ml of 1% SDS and incubate 15 min at 75°C.
This unhydrolyzed control will be used for evaluation of free amino groups in the raw material.
4. Add enzyme to the remaining substrate solution and stir mixture throughout the course of the reaction.
Biochemical Compositional Analyses of Proteins
5. Take 0.25-ml samples at suitable time intervals during the reaction. Immediately transfer each sample to a test tube containing 2 ml of 1% SDS and keep at 75 °C in a shaking water bath for 15 min.
This inactivates the enzyme and disperses the protein hydrolysate.
6. Transfer 0.25 ml from each sample (step 5) and control (step 3) to separate 16 x 125-mm glass test tubes containing 2 ml of 0.2 M sodium phosphate buffer.
7. Prepare six to ten calibration standards by diluting 2.5 mM leucine standard containing 0.25 to 2.5 amino meq/g in sodium phosphate buffer at a total volume of 2.25 ml.
As with the samples, the standards and blank should be prepared in appropriate replicates.
8. Prepare a blank containing 0.25 ml of 1% SDS in 2 ml sodium phosphate buffer.
9. Add 2 ml of 0.1% TNBS solution to each tube, vortex, and incubate in the dark at 50°C for 60 min.
10. Stop the reaction by adding 4.0 ml of 0.1 N HCl. Allow samples to reach room temperature and read absorbance at 340 nm.
11. Create a calibration curve by plotting A340 versus concentration of the standards.
12. Determine the degree of hydrolysis (see Basic Protocol 1, steps 12 to 14).
ALTERNATE PROTOCOL 2
Evaluation of the Progress of Protein Hydrolysis
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