Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for protein structure determination
Enzymatic hydrolysis

Prior to enzymatic Hydrolysis, the protein sample must be denatured and its Cysteine residues converted into hydrophilic derivatives. To this end, Disulfide Bonds in the protein are first reduced with dithiothreitol [37], and the cysteines are modified with iodoacetic acid (using [¹⁴C]-labeled iodoacetic acid for radioactive tagging), iodoacetamide [31], or ethylenimine [66], or oxidized with performic acid [30]. The choice of reagent is determined by the overall research strategy. Modification reactions are discussed in greater detail in Chapter 2.

Treatment with performic acid oxidizes not only cysteine residues but also Methionine and Tryptophan residues, and generally yields more soluble products than reductive alkylation. Aminoethylation of cysteine residues is used to introduce additional bonds into the protein that are susceptible to Hydrolysis by Trypsin or Armillaria mellea protease.

Ideally, proteolytic Cleavage should result in each region of the polypeptide chain being represented in the hydrolyzate by a single unique peptide generated in the highest possible yield. In practice, this ideal is rarely achieved because each protease exhibits varying degrees of affinity for different Regions of the polypeptide chain and cleaves bonds at varying rates. However, the proteolysis can be restricted to sites of highest affinity. Generally, this is accomplished by decreasing the Enzyme-to-substrate ratio or by increasing the volume of the added buffer solution; shortening the hydrolysis time also increases the yield of fragments produced by rapid Cleavage at the most “active” sites. “Mild” Protein Hydrolysis conditions for several Proteolytic Enzymes are summarized in Table 10.3. Proteolytic cleavage can be monitored using a pH-stat [18]: the cessation of acid or base consumption indicates that the reaction is complete.

Class="center">Table 10.3. Conditions for protein hydrolysis by various proteases (37 °C)^a

^a Protein-Substrate Concentration of 1 mg/mL in 0.5% (w/v) ammonium bicarbonate, pH 8.0. Similar conditions can be used for the hydrolysis of purified Peptides (1 µmol/mL). Peptide fragmentation conditions for Thermolysin: 1/100, 60 °C, 2 h; for Pepsin: 1/100, 37 °C, 4 h in 5% formic acid.

The “nonspecificity” of cleavage by a given enzyme may be caused by the presence of other protease impurities. For example, trypsin may contain chymotrypsin contamination, which can be removed by Affinity Chromatography, whereas tryptic activity in chymotrypsin is suppressed by adding a trypsin inhibitor prior to hydrolysis.

Denatured protein is typically insoluble in the ammonium bicarbonate buffer used for proteolysis and must first be solubilized or at least converted into a fine suspension. To achieve this, the lyophilized protein preparation is dissolved in a minimal volume of a denaturing agent (6 M guanidine hydrochloride, 8 M urea, 100% formic acid, or concentrated aqueous ammonia) and diluted with a significantly larger volume of buffer solution. Hydrolysis with trypsin, chymotrypsin, or elastase can be carried out in a 2 M urea solution [24], and with staphylococcal protease in 4 M urea [15]. If formic acid or ammonia was used to dissolve the protein, the buffer solution is basified with ammonia or acidified with formic acid to pH 8, respectively (since ammonium formate is a volatile compound, subsequent Desalting is unnecessary). The Use of urea and, in particular, guanidine hydrochloride requires desalting at one of the final stages.

The progress of proteolysis in suspension can be monitored by measuring the absorbance of the generated peptides in a small supernatant sample (after sediment removal) at 280 nm or by determining the radioactivity of [¹⁴C]carboxymethylcysteine. Completion of hydrolysis is judged by the disappearance of the intact protein band in a polyacrylamide gel. During the analysis, the bulk of the hydrolyzate is stored at –20 °C, and if Digestion is incomplete, the sample is thawed and proteolysis is continued. At The final stage, insoluble peptides are separated by centrifugation, and both portions of the hydrolyzate are lyophilized. It is advisable to subject the insoluble peptides to repeated hydrolysis with a higher enzyme load and combine the resulting peptides with the main batch (see also Chapter 3).