The binding of acidic dyes and other negatively charged ions is a well-known property of the proteins of blood plasma and is a specific property of the albumin fractions, the globulin fractions being almost inactive in this respect. This is easily observed when an acidic dye such as bromophenol blue is added to plasma and the plasma then subjected to electrophoresis on filter paper. The blue dye travels solely with the plasma albumin, leaving the globulin fractions unstained. A similar result is observed with the naturally occurring bile pigments in jaundiced plasma. Much further evidence is available for this specific property of plasma albumin and has been reviewed by Goldstein (1949), by Klotz (1949), and by Edsall (1947). Smith and Smith (1938) showed that for a number of plasmas from pathological as well as normal states the retention of phenol red by an ultrafiltration membrane was closely related to the albumin content of the plasma. Rosenfeld and Surgenor (1952) have proposed a method of estimation of human plasma albumin in whole plasma by using the interaction with haematin, the extent of the interaction being measured spectrophotometrically.In the present paper, the substance 1-anilinonaphthalene-8-sulphonic acid is used to estimate the dye-binding capacity of human plasma from a number of pathological conditions. The substance is non-fluorescent in aqueous solution, but brightly fluorescent when absorbed by plasma albumin and is one of a class of compounds with this unusual property described elsewhere (Weber and Laurence, 1954). The method to be described depends on the use of a limited amount of plasma, so that the fluorescent substance is always present in excess of the dye-binding capacity. The amount of dye bound is then a measure of the dye-binding capacity of the plasma and it is evaluated from the fluorescent intensity as only the bound dye is fluorescent. By using standard solutions of a purified albumin to relate albumin concentration with dye-binding capacity, the fluorescent intensities may be converted to equivalent albumin concentrations. Comparisons between these estimates and results of independent determinations by standard techniques are given. As no generally accepted absolute method of albumin determination in plasma is available, the best criterion of the validity is statistical agreement between any two independent methods. This criterion is used to assess the fluorimetric methods and also the standard methods of albumin determination. Fluorimetric MethodsReagents.-The following reagents were used in the fluorimetry:Fluorescent Standard Solution.-l-Naphthylamine-6(7)-sulphonic acid (B.D.H.), 40 mg., was dissolved in 100 ml. of 0.1 M-borate buffer (pH 9.2) and diluted to 1 litre with water.Stock Dye Solution.-l-Anilinonaphthalene-8-sulphonic acid was prepared by the method of Hodgson and Marsden (1939) and 4 mg. was dissolved in an equivalent amount of 0.lN-NaOH and diluted to 500 ml. with water.The l-anilinonaphthalene-8-sulphonic acid was prepared by heating together for 15 ho...

The results obtained here consistently failed to show deviations from Beer's law for optical densities less than 1.4, and the use of scanning as a convenient method for differential protein estimations would appear to be justified. The methods described may enable other workers to make similar tests of the method with a minimum of preliminary development, and some may succeed in obtaining significant failure of Beer's law in their apparatus. The use of apparatus of this type would enable the variation of the deviations with arrangement of the optical system, etc., to be worked out, but the following experimental conditions are already known to lead to significant deviations: 1. Use of dry paper instead of lightly oiled paper. 2. An inadequate light ifiter for the photocell. 3. An illuminated slit too long to be completely covered by the protein pattern. 4. Variations in protein density over the slit, either because the slit is too wide or because the protein pattern has been applied unevenly. 5. Use of low quality ifiter paper containing "pin holes." 6. A dye uptake which is not proportional to protein content of the paper [Martin and Franglen (14)]. The failure of Crook, Harris, and Warren (4) to substantiate Beer's law does not indicate the most general situation for the application of the scanning method. The application of Beer's logarithmic law to the scanning of dyed protein patterns has been investigated by methods described in detail. No deviations could be found for optical densities less than 1.4 for amidoschwarz 1OB or less than 1.1 for azocarmine B staining. The scanning method can be used for evaluation of protein fractions if care is taken.

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