Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Phosphorylated Amino Acids
Protein Hydrolysis

Phosphoester bonds in O-phospho derivatives of Serine, Threonine, and Tyrosine are cleaved by strong acids; therefore, short Hydrolysis times must be employed to prevent their degradation [322]. This acid lability also accounts for the lack of a reliable quantitative METHOD FOR DETERMINING phosphoamino acids in Proteins. The detection of Amino Acids containing a radioactive label [³²P] is carried out by autoradiography or by measuring radioactivity using a counter.

Phosphorylated Peptides can be separated from non-phosphorylated ones and fractionated by HPLC under isocratic conditions at 22 °C on a Spherisorb C₁₈ Column using a phosphate buffer (pH 3.2–4.5) and n-hexanesulfonic acid as a counterion [114]. About 0.1 nmol of peptide can be identified by absorbance at 210 nm; The Use of a fluorescent or radioactive label [³²P] increases sensitivity 100-fold.

The protein is treated with 6 M HCl at 110 °C for 1–4 h. The hydrolyzate is filtered through a Millipore filter (0.22 µm), and the HCl is removed in vacuo. The residue is dissolved in 0.001 M TFA, and unlabeled Amino acids are added as standards [51].

Phosphohistones (100 µg) are hydrolyzed with 6 M HCl in vacuo at 100 °C for 4 h or by incubation with 100 µg of protease from S. griseus in 50 mM Tris–HCl buffer (pH 7.5) at 20–24 °C for 20 h [358].

Enzymatic hydrolysis of [³²P] Phosphoproteins. Dissolve the protein in 100 µL of 0.1 M ammonium bicarbonate containing 2 mM EDTA (neutralized) and 250 nmol of each unlabeled phosphoamino acid: Ser(P), Thr(P), Tyr(P). Add Trypsin (200 µg/mL) and incubate for 12 h at 37 °C in the presence of EDTA, which inhibits any phosphatase activity introduced with the protease. Then, add aminopeptidase M (200 mU) and incubate for an additional 12 h at 37 °C (J. Downward, 1984, personal communication).