Protein Protocols Protein Protocols

466 Tawfik
reagent. Dramatic pK a changes are often found in active sites; thus, certain residues
might be particularly difficult to modify, thereby forcing conditions that result in a
nonspecific loss of activity.
A number of quite simple experiments may strongly support the results obtained by
chemical modifications:
1) A simple control that allows the modification to be ascribed to the active site of a protein
is to demonstrate protection (i.e. lack of modification) in the presence of a specific ligand
to that site, for example, a hapten for an antibody, a substrate or an inhibitor for an enzyme
(for an example see Chapter 64).
2) The extent of modification is determined primarily by the molar excess of the modifying
reagent, but also by other conditions such as pH, temperature, and reaction time. Reliable
and reproducible results are generally obtained only after a wide range of reagent concentrations
are applied under different reaction conditions. Following these modifications,
one should determine not only the remaining biological activity but also, when possible,
the number of modified side chains (details for each reaction are provided in the following
chapters). These data may allow one to assess to what extent the modification is indeed
site specific (e.g., loss of activity is the result of modification of one or two amino acids of
the type modified) or whether loss of activity is due to a complete disruption of the protein
due to, for example, the modification of a large number of amino acids.
3) Some of the modifications are reversible; for example, histidine side chains modified by
diethyl pyrocarbonate can be recovered by a short treatment with hydroxylamine. Recovery
of the activity of the modified protein following this treatment may demonstrate the
specificity of the modification (see Chapter 65). Additional reversible modifications are
described in Chapters 68 and 70.
4) Demonstrating pseudo-first-order kinetics for the inactivation of the protein may indicate
that the modification proceeds like an ordinary bimolecular reaction and not via the formation
of a binding complex as with affinity labelers or suicide inhibitors.
In the following chapters I have provided basic protocols for the specific modifications
of different side chains; these, or very similar, protocols can be applied with other
reagents as well (see refs. 1–4). The first protocol for the nitration of tyrosine side
chains with tetranitromethane (Chapter 64) is written for a specific protein (an antidinitrophenyl
[DNP] antibody) and provides as many experimental details as possible.
Hence it is recommended to read Chapter 64 (including the Notes) before applying any
of the other modifications described in Chapters 65 to 70.
1.1. General Notes
1. To avoid misleading results it is important to be familiar with the chemistry of each of the
reagents (see refs. 1–4 and references therein). A detailed mechanistic discussion is
beyond the scope of this book, although examples for typical problems or side reactions
are given in each chapter.
2. Many of the reagents and solvents described are harmful and should be used only in a
well-ventilated hood and while applying other precautions such as wearing suitable gloves.
3. Examine the buffer you intend to use with your protein in light of the modification reaction.
For example, while amidating carboxylate groups (see Chapter 67), the use of an
acetate buffer, or of any other buffer that contains carboxylate groups or other nucleophiles
(e.g., Tris buffer), should obviously be avoided. Likewise, the use of certain organic solvents
should be avoided (e.g., acetone with 2-hydroxy-5-nitrobenzylbromide, see Chapter 69).