Protein error

The effect of proteins on the dissociation constant of indicators (often called protein error) is well known. It is defined as the difference between the colorimetrically determined values of pH in the presence and in the absence of the proteins in solution, whose pH has been adjusted (usually electrochemically) to its original value. Depending on the type of proteins, their concentration, and on the type of the indicator, it can be as high as 0.8 units of pH. Again, it has its origin in the specific interactions between the indicator molecule and the proteins. 

A second possibility is to divide the fractions into the albumin on one side and the globulins on the other and to apply one single correcting factor to the albumin. This factor is a compound of the protein and of the paper factor. Protein and paper errors are most important for the large and concentrated albumin fractions. Fortunately they partly compensate each other, the paper error tending to lower the albumin value and the protein error tending to raise it. We use 1.5 in our own experimental setup and multiply the amido black-albumin surface by this figure. 

It has been well known for a relatively long time that micellar, i.e. association colloidal, systems have a considerable effect on such indicator equilibria. Indeed, in the 1920 s and early 1930 s experiments were carried out in order to elucidate the so-called colloid or indicator error (Hartley, 1934 Hartley and Roe, 1940). In addition, the protein error was noted in investigations involving acid-base titrations in the presence of proteins (Sorensen, 1929 cf. Hartley, 1934). These errors are, of course, the consequence of micellization and the subsequent effects of micelles on equilibrium (34). The importance of many indicators in the dye, textile, and photographic industries, and the analytical utility of the shifts in indicator equilibria prompted much of the research in this area. 

E532 Kulpmann, W.R. (1989). Influence of protein on the determination of sodium, potassium and chloride in serum by Ektachem DT 60 with the DTE Module Evaluation with special attention to a possible protein error by flame atomic emission spectrometry and ion-selective electrodes proposals to their calibration. J. Clin. Chem. Clin. Biochem. 27, 815-824. 

The dipstick test for total protein includes a cellulose test pad impregnated with tetrabromphenol blue and a citrate pH 3 buffer. The reaction is based on the protein error of indicators phenomenon in which certain chemical indicators demonstrate one color in the presence of protein and another in its absence. Thus tetrabromphenol blue is green in the presence of protem at pH 3 but yellow in its absence. The color is read after exactly 60s and the test has a lower detection limit of 150 to 300mg/L, depending on the type and proportions of protein present. The reagent is most sensitive to albumin and less sensitive to globulins, Bence Jones protein, mucoproteins, and hemoglobin. 

The properties of most of these indicators are known only incompletely (purity, salt error, protein error), and they must be employed with great care. It is advisable for practical purposes to select a list of indicators which have proven to be most useful. Such a list is found in the accompanying table. 

Phenolphthalein is a very useful indicator. Its protein error is quite small. 

Commercial preparations may be purified by adding hydrochloric acid to an aqueous solution in order to precipitate the indicator, which is then washed and dried. A small quantity of alkali, insufficient to dissolve all of the indicator, is added and the solution evaporated to yield the sodium salt. A 0.1% aqueous stock solution is prepared, and 1-3 drops used per 10 c.c. of solution under investigation. The interval occurs at pH 1.3-4, the color going from blue-violet to red. This substance has a high salt error and protein error, and is not recommended for use as an indicator. 

The purification of commercial samples and preparation of a stock solution is the same as for benzopurpurin. The interval lies between pH 3.0 and 5.2, the color changing from blue-violet to red. This compound likewise has high salt and protein errors, and thus is not a very desirable indicator. 

Litmus (azolitmin) is the classical indicator for acid-base titrations, although by now it has been supplanted by much better indicators. Neither litmus nor azolitmin should be used in colorimetric determinations of pH because of their salt and protein errors. Litmus is of value only in the form of indicator paper (cf. Chapter Eleven). 

The protein error depends also upon the indicator involved. According to SSrensen the error is smaller the simpler the composition of the indicator. Whenever a given indicator is used in protein-containing solutions, it is advisable to check several of the results with a hydrogen electrode. Reliable data may be expected if the agreement is good. 

Sven Palitzsch showed that the protein error of methyl red was very small. This conclusion was drawn from the following experiments with a 2% solution of natural egg white in dilute hydrochloric acid solution. 

B. Cohen Public Health Reports, 4, 3051 (1926). Cf. D. Jaumain Compt, rend. soc. biol., 03, 860 (1925), regarding the protein error of bromthymol blue. 

In cases where the protein error is not known in advance, it is advisable to determine the pH both with an indicator acid and an indicator base. The result may be considered reliable if both measurements agree. 

When we are no longer dealing with an indicator in true solution, certain additional difficulties enter due to the difference in behavior of the two indicator forms at an interface. The protein error results in part from a colloidal behavior. 

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