Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Dicarboxylic amino acid amide residues
Methods for Amide Determination

Under standard acid Hydrolysis conditions (6 M HCl), asparagine and glutamine are converted into aspartic* and glutamic acids, respectively. The total amount of dicarboxylic amino acid amides can be determined from the quantity of ammonia released under selective acid hydrolysis conditions (concentrated HCl at 37 °C for 10 days, or 2 M HCl at 100 °C for 2–8 h). Simultaneously, a slight deamination of Serine, Threonine, cystine, and Tryptophan residues takes place. A portion of the hydrolysate is alkalized in a Conway microdiffusion apparatus, and following overnight diffusion, ammonia is quantified colorimetrically via the ninhydrin reaction [401]. Glutamine undergoes deamidation more readily than asparagine [32]. The amides of aspartic and glutamic acids can be determined separately using chemical and enzymatic Methods [369]. See review [8].

Even after dialysis, Proteins frequently contain free ammonia, which is removed by passing the protein solution through a Column packed with a cation-exchanger in the H⁺-form.

8.8.1.1. Diffusion Method [401].

Control experiment. Dissolve 10 mg of protein in 1 ml of 0.03 M HCl in methanol and precipitate with 2 ml of ethyl ether. Centrifuge. Repeat the Procedure twice. Dry the precipitate in vacuo and quantitatively transfer it to the outer chamber of a Conway diffusion Cell. Place 1 ml of 0.02 M H₂SO₄ into the central compartment of The Cell. Quickly and carefully add 1 ml of a saturated sodium tetraborate solution (alkalized to pH 10.5 with 2 M NaOH) to the protein precipitate. The vessel must be hermetically sealed with an additional weight placed on top. The diffusion process proceeds overnight at room Temperature. Quantitatively transfer the Contents of the central compartment into a flask and dilute with Water to a predetermined volume. Determine ammonia by the ninhydrin photometric assay.

Amide nitrogen in protein. Incubate 2 mg of protein in 1 ml of 2 M HCl at 100 °C for 2–8 h; cool and, if necessary, centrifuge. Transfer 100–250 µl of the solution into the outer chamber of the Conway cell, as in the control experiment, but using a more concentrated borate buffer (15 mg of boric acid in 100 ml of 2 M NaOH). Each measurement requires ~0.2 µmol of NH₃. Plot The amount of released NH3 against time and determine the intercept value by extrapolation to zero time.

8.8.1.2. Diffusion method using trinitrobenzenesulfonic acid [399].

Reagents.

2,4,6-Trinitrobenzenesulfonic acid (TNBS) recrystallized from 1 M HCl in water [110].

TNBS solution (1.1 M; 100 mg/200 ml of water) is prepared fresh. Store frozen in the dark.

Ammonium sulfate (analytical grade, Merck) dried for 3 h at 100 °C and stored in a desiccator over silica gel.

Boric acid, 0.1% solution (w/v).

Na₂B₄O7, saturated aqueous solution.

A. 0.1 M Na₂SO₃; prepare daily.

Б. 0.1 M NaH₂PO₄.

B. 0.1 M Na₂B₄O₇ in 0.1 M NaOH.

Г. 1.5 ml A + 98.5 ml B (prepare daily).

Procedure. Hydrolyze the protein with 10 M HCl at 37 °C for 10 days. Place the hydrolysate into the outer chamber of a Conway diffusion cell and add water to bring the total volume to 1.5 ml. Pipette 0.9 ml of 0.1% boric acid into the central compartment. Apply stopcock grease to the chamber lid, lift it slightly, and rapidly add 3 ml of saturated sodium tetraborate solution adjusted to pH 10.5 with 2 M NaOH. Seal the chamber tightly and place a weight on top of the lid. Leave the reaction mixture for 20 h at room temperature, then quantitatively transfer the contents of the central compartment to a tared microcentrifuge tube and add water to bring the solution mass to 1.5 g. This volume yields three samples for analysis According to the Fields assay [110] described below; the sensitivity is 6 nmol. Simultaneously determine the water and ammonium sulfate content.

Fields procedure. Add the sample to 0.5 ml of reagent B and adjust the volume to 1.0 ml. Add 0.02 ml of TNBS solution and mix thoroughly. After 5 min, terminate the reaction by adding 2 ml of reagent Г. Measure the absorbance at 420 nm.

8.8.1.3. Amino acid analyzer [173]. Hydrolyze 2–3 mg of protein in 1 ml of constant-boiling hydriodic acid containing 0.03% hypophosphorous acid (BDH-MAR); perform the hydrolysis in a sealed evacuated ampoule at 107 °C (forced-air incubator) for 1.5 h for soluble proteins and 2 h for insoluble proteins. Alternatively, other conditions may be used: 2 ml of 2 M HCl, 110 °C, 2 and 14 h. Dilute the hydrolysate with 5 ml of 0.1 M HCl and apply it to a column (75×9 mm) packed with Aminex A-5 resin using a Beckman amino acid analyzer. The actual amount of ammonia generated from amide groups is determined by subtracting the corresponding peak values obtained in a blank run (without Protein Hydrolysis) from the measured value.

Thus, using 1.5 mg of protein for hydrolysis and 2.5 mg for the control run, a single dicarboxylic amino acid amide residue was determined in a protein with a Molecular Weight of 7000. Hydrolysis with hydriodic acid and the micro-Kjeldahl method yielded comparable results. The top of the analytical column should feature a 1 cm layer of resin resting on a Teflon gasket to retain unhydrolyzed protein. This top portion of the resin (along with the gasket) is removed prior to loading the NaOH solution onto the column and is subsequently regenerated by heating at 70 °C with 12 M sulfuric acid.

8.8.1.4. Bis(1,1-trifluoroacetoxy)iodobenzene [345].

Reagent

Bis(1,1-trifluoroacetoxy)iodobenzene (BTI) can be prepared according to the procedure described in [287]. Dissolve 5.0 g of phenyliodosylate (Aldrich) in 25 ml of anhydrous TFA with gentle warming and let stand at room temperature for 1 h. Remove the solvent in vacuo. Recrystallize the residue from a hexane-TFA mixture. Melting point 123–126 °C. Store in a dark Glass bottle at −20 °C.

Procedure. Cleavage of carboxyamide groups. A solution of 200 µg of protein in 2 ml of 0.01 M TFA or 5 M guanidine∙HCl – 0.01 M TFA is mixed with 2 ml of freshly prepared BTI in dimethylformamide (36 mg/ml). Evacuate and seal the ampoule; heat at various temperatures (35, 60, and 110 °C) for increasing time periods (4 and 18 h). Remove excess reagents by dialysis against water (Spectrapor 3 membrane). Extract the dialyzed sample three times with equal volumes of n-butyl acetate. Lyophilize. Hydrolyze with 6 M HCl in vacuo at 110 °C for 24 h.

Asparagine is quantitatively converted into 2,3-diaminopropionic acid, and glutamine into 2,4-diaminobutyric acid. Since these acids are typically not resolved from Lysine on an amino acid analyzer, the amide content in the protein is determined by a differential method—namely, by measuring the Asp and Glu content in samples both before and after BTI Treatment. Certain Amino Acids (Cys, His, Lys, Met, and Thr) are affected during the reaction with BTI.

8.8.1.5. Enzymatic hydrolysis and Gas-Liquid Chromatography [150].

Enzymatic hydrolysis. A 0.2 µmol sample of peptide in 0.2 mL of 0.05 M NH₄HCO₃ (pH 8.0) is hydrolyzed with pronase (1% by weight) for 24 h at 37 °C. Hydrolysis is then continued for an additional 17 h by adding 4% (by weight) aminopeptidase M. The mixture is lyophilized.

Gas-liquid chromatography. The amino acid mixture in a vial with a Teflon-lined screw cap is treated with 0.2 mL of 1.25 M HCl in n-butanol at 100 °C for 7 min. The reaction mixture is cooled in ice and dried under a stream of nitrogen. Acylation is then performed by adding 0.1 mL of trifluoroacetic anhydride in methylene chloride (1:3) to the residue and heating the mixture for 20 min at 100 °C. The Amino Acid Derivatives are separated on a column (1.5 m × 4 mm) packed with Chromosorb W. n-Butyl stearate containing 0.325% (by weight) Ethylene glycol adipate is used as an internal standard. The specified Esterification conditions must be strictly observed to prevent the potential partial conversion of amides into aspartic and glutamic acids.