Albumin determination by dye binding

All proteins, and especially albumin, tend to react with many chemical species by means of electrostatic and tertiary van der Waal's forces and by virtue of hydrogen bonding. Bilirubin, fatty acids, most hormones, and many drugs are transported about the body bound to albumin. Many colored dyes in the anionic form also possess this protein-binding property. This property has been used in attempts to devise methods by which albumin could be measured directly, without previous precipitation of globulins.

In dye-binding methods, only those dyes or indicators can be used that bind very tightly to the albumin molecule, so that practically 100 per cent of the albumin present is bound to dye. The binding must be unaffected by reasonably small changes in ionic strength and pH. Further, the color of the protein-bound dye should be different from that of the free dye; i.e., there should be a substantial shift in the wavelength of light at which maximum absorption occurs in the two forms. The albumin-dye concentration can then be measured in the presence of excess dye. Finally, dye binding to other protein fractions (globulins) must be negligible if the dye binding is to be the basis for a valid assay of albumin. For practical reasons, the color characteristics of the dye should be such that it can be measured at wavelengths of light where bilirubin and hemoglobin will give negligible or minimal interference. The use of methyl orange was proposed by Bracken and Klotz and Watson and Nankiville; HABA (2-(4'-hydroxyazobenzene)-benzoic acid) by Rutstein, Ingenito, and Reynolds and Martinek, and bromcresol green by Rodkey. The use of bromcresol green is most free of pigment interference. Bilirubin at levels over 5 mg./100 ml. interferes with HABA procedures and to a lesser degree with methyl orange methods.

The use of these dye reagents is now well established, and several manual and AutoAnalyzer procedures using methyl orange and HABA are available; however, there is considerable disagreement over the reliability of the dye-binding methods. Occasionally, albumin values obtained are unusually high as a result of the binding of the dye to proteins other than albumin. As an example of dye-binding technique, a methyl orange procedure will be presented in outline. It is a modification of the method proposed by Wrenn and Feichtmeir and is designed for use with the Coleman spectrophotometer and 19 mm. cuvets.

Assay of Serum Albumin by Methyl Orange Binding
Reagents

1.  Working dye reagent. Prepare by adding 5 to 10 ml. portions of 0.10 per cent (w/v) methyl orange to 1000 ml. of citrate buffer, 0.055 M. pH=3.50 +/- 0.03; check the absorbance against water at 540 nm. after each addition. Continue addition of dye until the absorbance lies between 0.78 and 0.85. Use the reagent at 24 to 27 degree of Celcius, although it is stored in the refrigerator.
2. Standards. These are prepared from crystalline human serum albumin. Bovine albumin should not be used because there are significant differences in dye binding by albumins from different species. It is wise to assay the albumin standard against the biuret total protein method so that the total protein and albumin values will be consistent. Prepare the stock reagent, which has a concentration of 10.00 gm./100 ml., by dissolving the albumin in saline. Prepare the working standards by diluting the stock with saline to give levels of 1.0 through 6.0 gm./100 ml.

Procedure

1.  Pipet 10.0 ml. of working dye reagent into a series of cuvets. One cuvet will be needed for the albumin blank, one each for the six standards, and one for each of the unknowns. Transfer 0.20 ml. each, of saline, standards, and sera, to the respective cuvets. After mixing by gentle inversion, permit the cuvets to stand for 20 minutes.
2. Read the absorbances of the standard and serum specimens against the blank at 540 nm. If the specimens are slightly lipemic, icteric, or hemolyzed, pigment color corrections will have to be made. For this purpose, add 0.20 ml. of the test serum to 10.0 ml. of the citrate buffer and read the absorbance against water (or citrate buffer) at 540 nm. Subtract these readings from the previous readings.
3. Convert the absorbance readings to albumin concentration by use of the calibration curve, which follows Beer's law up to concentrations of about 4 to 5 gm./100 ml. Prepare new calibration curves daily.

 Comments
Normal globulins and most abnormal globulins have no effect on the albumin values. Occasionally paraproteins and macroglobulins will interfere by giving high results. Dye binding, being a dissociation-association equilibrium phenomenon, is influenced by changes in temperature; a rise in temperature increases the dye-albumin dissociation and thus gives lower results. Therefore, standards and sera should be run under identical conditions. Heparinized plasma should not be used because heparin, for reasons not understood, enhances dye binding by albumin. Serum albumin levels obtained by methyl orange dye binding techniques tend to be 0.1 to 0.2 gm./100 ml. higher than those obtained by salt-fractionation or electrophoresis, although some chemists have reported results as much as 0.5 gm./100 ml. higher.