Quantitative Determinations of Proteins

The quantitative estimation of proteins is one of the basic requirements

in biochemistry. In reviewing the biochemical literature for

methods of fast and sensitive determination of the amount of protein,

the large variety of proteins becomes evident, since theamount

of protocols for quantitative protein seems to be innumerable.

Proteins, from many points of view, are much more complex

than, for example, nucleic acids. As a result, it has been difficult to

give laboratory protocols that can be applied to proteins in general;

however, in most cases the specialized protocolsmay be reduced to

a few basic methods. But if a protein becomes pure or some of its

unique properties are of special interest, another analytical method

has to be used. Nevertheless, accurate quantitation of the amount

of protein during the steps of protein preparation is the only valid

way to evaluate the overall value of a procedure.

The following protocols are based on distinct properties of

proteins; therefore, exact information is only possible if a heterogeneous

protein mixture is compared with a universal standard

protein. The best way would be to take a defined sample of the

protein to be analyzed. So the difficulties start with the selection of

the standards, because it is well known how difficult it is to prepare

a protein that fulfills the criteria of analytical chemistry.

It is very often observed that during a purification process the

differences increase between the real amounts of a protein and the

values obtained by any method, e.g., total enzyme activity, because

the measured signal produced by a protein mixture differs from

that of a pure protein. Furthermore, the amount of a given protein

determined by a distinct protocol differs fromthe expected amount

by portioning, as shown in Table 1.1. To avoid additional mistakes

with the already uncertain process, the protein estimation method

should not be changed during a purification process.

With these difficulties kept in mind, any protein may be estimated

by one of the given protocols. Absolute statements, such as

“… the prepared, pure product has a specific activity of … units

per milligram of protein …” should be made with caution.