Overview of Hydrolysis Methods

How to Perform Vapor Phase HCl Hydrolysis*

  1. Vacuum dry sample in clean 6 x 50 mm Pyrex tube. Use ~1 痢 for PTC-AAA and 5-10 痢 for ninhydrin-AAA.
  2. Place sample tubes in a 40 ml screw cap bottle containing 200 無 6N HCl plus a crystal of phenol. Cap with a modified mini-inert slide valve (Pierce).
  3. Evacuate and flush with argon 3 times. Close slide valve on last vacuum step.
  4. Heat 1 h at 150C. Following hydrolysis release pressure within a fume hood, pointing away from your face!
  5. Transfer sample tubes to another container and vacuum dry. Store at -20C under argon until submitted for AAA.

* from G.E. Tarr (1986) In: Microcharacterization of Proteins (J.E. Shively, ed.), Humana Press, pp. 155-194.

How to Perform Liquid Phase HCl Hydrolysis*

  1. Vacuum dry sample in clean glass test tube (10 x 150 mm).
  2. Add 100 無 6N HCl containing 4% thioglycolic acid directly to the sample.
  3. Evacuate the hydrolysis tube to <100 microns and flame seal.
  4. Heat 22 hours at 110C.
  5. Open cooled tube, vacuum dry and analyze.

* M.C. Miedel, J.D. Hulmes and Y.-C.E. Pan (1989) J. Biochem. Biophys. Methods 18, 37-52.

Hydrolysis Method for Tryptophan Analysis

ABRF has compared several tryptophan methods. (1)

Performance of Tryptophan Methodolgy, ABRF-91AAA and -92AAA Combined
Method Total SitesSites with <10% Trp Error
Dodecanethiol116 (55%)
Methane Sulfonic Acid174 (24%)
Mercaptoethane Sulfonic Acid53 (19%)
Thioglycolic Acid161 (20%)
Other70
Total5614 (25%)

Autohydrolysis with Dodecanethiol in PTC-AAA (2)

  1. Apply 40 無 40% dodecanethiol in heptane to sample frit of ABI Model 420H, 10-15 minutes before sample application.
  2. Apply 0.5 - 2.0 痢 of sample and carry out normal autohydrolysis (1h,165C with 6N HCl).
  3. Run control proteins such as a-Lactalbumin and Lysozyme to calculate accurate response factor for unknown samples.

Hydrolysis with 4N Methane Sulfonic Acid (3)

  1. Add 50 無 4N methane sulfonic acid to dried sample in vial.
  2. Seal tube in vacuo and heat 22 hours at 110C.
  3. Neutralize with 50 無 4N NaOH.
  4. Analyze on post-column instrument.

1 D.J. Strydom et al. (1993) In: Techniques in Protein Chemistry IV (R.H. Angeletti, ed.) Academic Press, pp. 279-288.
2 Bozzini et al., Applied Biosystems Research News, February 1991; K.A. West and J.W. Crabb (1992) In: Techniques in Protein Chemistry III (R.H. Angeletti, ed.) Academic Press, pp. 233-242.
3 B.N. Jones et al. (1981) J. Liquid Chromatography 4, 565-586.

Phosphoamino Acid Analysis

  • Hydrolysis in 6N HCl for 1-4 h at 110C allows semi-quantitative analysis of PSer, PThr and PTyr using either pre-column or post-column systems.
  • Warning: Phosphoamino acid analysis can be time consuming and yield disappointing results. This is not a routine analysis in most facilities.
  • Recommendation: Develop expertise in standard amino acid analysis before pursuing phosphoamino acid analysis.
  • No single method allows quantitative analysis of phosphoamino acids and all 16 common amino acids in protein hydrolysates.
  • Chromatography adjustments are typically required to optimize resolution of phospho-amino acids from other residues.
  • For a recent overview of core facility phosphoamino acid analysis, see Yuksel et al., (1994) Techniques in Protein Chemistry V (JW Crabb, ed.) Academic Press, pp. 231-240.

Other Hydrolysis Methods

Microwave Hydrolysis (1)

  • Very fast (<10 minutes)
  • Special equipment needed
  • Appears to work well

Proteolysis

  • Complete proteolysis with a combination of proteases can sometimes be useful for identifying modified amino acids. This can be tricky and requires appropriate controls. Generally more useful with peptides than proteins.

Automated Hydrolysis (2)

  • Reduces operator variability and labor
  • Enhances accuracy below 1 痢 hydrolyzed.
  • Benefits outweigh the maintenance requirements.

1 L.B. Gilman and C. Woodward (1990) In: Current Research in Protein Chemistry (J.J. Villafranca, ed.) Academic Press, pp. 23-36.
2 K.A. West and J.W. Crabb (1990) In: Current Research in Protein Chemistry (J.J. Villafranca, ed.) Academic Press, pp. 37-48.

Comparison of Automatic and Manual Hydrolysis

K.A. West and J.W. Crabb (1990) In: Current Research in Protein Chemistry (J.J. Villafranca, ed.) Academic Press, pp. 37-48.

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