EFFECT OF HIGH SALT CONCENTRATIONS ON COLOR PRODUCTION OF THE BIURET REACTION FOR PROTEIN ANALYSIS HAROLD L. ROSENTHAL, PH.D., AND TOYOKO KAWAKAMI, M.T. (ASC1>) Division of Biochemistry, Department of Pathology, The Rochester General Hospital, Rochester, New York The biuret reaction for the determination of serum protein, introduced by Kingsley, 6 and modified by Weichselbaum, 11 Mehl, 8 and Gornall, Bordawill, and David, 8 is admirably suited to routine determinations in the clinical chemistry laboratory. The procedure requires appropriate standardization, however, in order to obtain reliable results. The use of the biuret reaction for the determination of levels of albumin in serum, after fractionation with sodium sulfite 1 or sodium sulfate, 4 7 9 may yield values that are consistently higher than those obtained with the classic micro-kjeldahl procedures. This effect differs from the turbidity that is produced by calcium salts, as previously described by Rosenthal and Cundiff. 10 A study of the biuret reaction indicated that high concentrations of salts alter the absorbance of the biuret color. The "salt effect" was observed by Gornall and co-workers 3 with sulfite and methanol, but not with sulfate. This paper deals with a more detailed study of the "salt effect." MATEKTALS AND METHODS Reagents 1. Biuret reagent. The biuret reagent proposed by Gornall, Bordawill, and David, 3 as described by Fister, 2 was used in this study. Place 1.5 Gm. of crystalline copper sulfate (CuS0 4 5 H2O) and 6 Gm. of sodium potassium tartrate (NaKCJLOe 4 H 2 0) in a 1-1. volumetric flask, add approximately 500 ml. of distilled water, and dissolve the salts. While constantly shaking the flask, add 300 ml. of 2.5 N sodium hydroxide, and then dissolve 1 Gm. of potassium iodide (KI) in the mixture. Dilute to the mark with distilled water, and store the solution in a polyethylene or ceresin-lined bottle. The reagent has a ph of 11.4, and it is stable almost indefinitely. All of the chemicals used in preparing the reagent should be of reagent grade. The 2.5 N solution of sodium hydroxide is made by diluting a saturated, carbonatefree solution, and then titrating with a standardized solution of hydrochloric acid. 2. Standard solution of protein. Human serum albumin (containing 25 per cent Received, March 27, 1956; accepted for publication June 4. Dr. Rosenthal is Chief of Biochemistry, and Mrs. Kawakami is Biochemistry Technician. 1169
1170 ROSENTHAL AND KAWAKAMI Vol. 26 proteins) was obtained from the American Red Cross.* This preparation was diluted with physiologic solution of sodium chloride, so that the final concentration was approximately 5 Gm. per 100 ml. The diluted solution of albumin was then standardized by means of micro-kjeldahl determinations, using selenium oxychloride as the catalyst. 3. Salt solutions. Concentrated solutions of neutral salts (Baker or Mallinckrodt analytic reagent grade) were prepared in distilled water, and the ph adjusted to 7. Concentrated solutions of phosphates were prepared in distilled water, and the reaction adjusted to the desired ph with phosphoric acid or an appropriate base. Procedures 1. Effects of salts. Appropriate quantities of salt solutions were added to 1-ml. aliquots of the standardized solution of albumin in a series of test tubes, and the volume in each tube was adjusted to 5 ml. by the addition of distilled water. Blanks were included in each series of tests, using 1 ml. of physiologic solution of sodium chloride in place of the aliquot of albumin. As the basis for comparison, the tests also included tubes with the standard solution of albumin, diluted with water, in place of the salt solutions. Five ml. of biuret reagent were added to each tube, and the contents mixed by inverting the tubes. After standing at room temperature for 30 minutes, the absorbancies of the solutions were read in a Klett-Summerson colorimeter, using filter No. 54. All of the determinations were performed in duplicate. 2. Comparison of biuret and Kjeldahl methods for determination of serum albumin. The actual content of serum albumin was determined by fractionating the serum with 26.86 per cent solution of sodium sulfate (ph 6.9, ± 0.1), according to the method proposed by Milne. 9 Globulins were removed by means of extracting with ether, as recommended by Kingsley. 6 In performing the analysis, 4 ml. of solution of albumin and 6 ml. of biuret reagent were mixed, and the absorbancies were compared with that of the standardized solution of albumin, treated in a manner identical with that used for serums. Aliquots of the specimens were removed in order to make determinations of protein nitrogen by the micro-kjeldahl technic, and the results were corrected for the content of nonprotein nitrogen in trichloracetic acid filtrates. RESULTS AND DISCUSSION The color of the biuret-protein complex was greatly affected by high concentrations of salts, as indicated by the data in Table 1. Most of the salts increased the intensity of color beyond that observed with the standard. On the other hand, some of the salts, such as lithium chloride, seemed to depress the intensity of the color, whereas others had little effect. Similar salts, such as the chlorides of lith- * Rod cross albumin contains approximately 6 per cent of globulin that is insoluble in 26.86 per cent solution of sodium sulfate. It is not necessary to remove this protein, inasmuch as the material dissolves in the alkaline biuret reagent, and yields comparable densities of color that are proportional to the concentration of protein in the material.
OcCl956 PROTEIN ANALYSIS 117.1 TABLE 1 EFFECT OF CONCENTRATION OF NEUTRAL SALT ON PRODUCTION OF COLOR IN THE BIURET REACTION* Sodium sulfite Saltf Sodium sulfate Sodium nitrate Lithium chloride Lithium sulfate Potassium nitrate Potassium chloride Sodium chloride Concentration in the Tube millimols 2.2 S.9 7.6 4.7 18.8 9.2 36.6 2.3 9.1 2.7 10.7 3.6 14.6 4.6 18.2 Change in Color} per cent + 13.8 +1S.S + 12.5 +2.0 +2.0 0-14.1 +2.6 +3.2 +0.6 + 1.2 + 1.9 +4.4 +1.9 +9.3 * Tests performed with 5 mg. of human serum albumin per tube, f All of the solutions of salts were adjusted to a ph of. } Corrected for the appropriate reagent blank. ium, sodium, and potassium, varied widely in their effect. It is possible that such effects may be the result of contaminating trace materials in the preparation of salts. High concentrations of buffer salts depressed the production of color by means of altering the ph of the reaction mixture (Table 2). This was apparent in those instances where the ph of phosphate buffers was 7. Adjustment of the ph of the phosphate buffers (to the same level as that in the biuret reagent) eliminated this effect, with the result that a small "salt effect" was observed. Although they are much less soluble than the corresponding potassium salts, the sodium salts of phosphoric acid were more effective in altering the color produced in the biuret reaction. The concentrated solutions of salts did not alter the shape of the absorption spectrum of the copper-protein complex in the visible region, although the absorbance at the maximal wavelength was modified. The absorption spectrum of serum albumin with ultraviolet light, however, remained unaltered with respect to shape or absorbance when the albumin was treated with physiologic solution of sodium chloride, or with concentrated solutions of sodium sulfite or sodium sulfate. The absorbancies of blank solutions that contained high concentrations of salts were altered from the values for the controls in a manner similar to that with solutions of protein. Thus, it seems that concentrated solutions of salts modify the activity of copper ions in the reaction. The micro-kjeldahl and biuret procedures were used to determine the content of total protein in 19 specimens of serum, and the level of albumin in 14 speci-
1172 ROSENTHAL AND KAWAKAMI Vol. 26 TABLE 2 EFFECT OF CONCENTRATION OF BUFFER SALT ON PRODUCTION OF COLOR IN THE BIURET REACTION* Buffer Salt Concentration in the Tube ph Change in Colorf Dipotiissium phosphulc Disodium phosphate millimols 10.9 10.9 O.S O.S 11.4 11.4 11.0 11.0 per cent -2.3 +3.1-22.0 -S4.1 +2.5-1.8 +2.4 +1.S -10.2 +7.8 * Tests performed with 5 mg. of human serum albumin per tube, t Corrected for the appropriate reagent blank. TABLE 3 COMPARISON OF METHODS FOR THE DETERMINATION OF SERUM PROTEIN Determination Number of Samples Kjeldahl Method Biuret Method Total protein Average Range Albumin* Average Range 19 14 Gm./lOO ml. 7.25 5.52 to 11.32 3 2.31 to 4.39 Gm./lOO ml. 7.29 5.68 to 11.47 4 2.3S to 4.39 'Determined after fractionation of serum with 26.86 per cent solution of sodium sulfate. mens that were fractionated with 2G.86 per cent solution of sodium sulfate. The comparative data are listed in Table 3. When the values were appropriately corrected for the "salt effect," the results from the biuret reactions were closely similar to those obtained with the micro-kjeldahl analyses. Such studies indicate (1) that the biuret reaction is a reliable procedure for determining the level of total serum protein, as well as serum albumin (after fractionation of the serum with sodium sulfate), and (2) that the biuret reaction under proper conditions yields results that are comparable with those of the classic, but lengthy micro-kjeldahl procedure. The availability of carefully standardized human or bovine serum albumin from commercial sources obviates the necessity of standardizing pools of human serum for use as standards. Thus, a standard sample of protein may be included with each test. If this standard sample is treated in a manner that is identical with that used for the serum to be tested, the analyst will have a precise and accurate means of evaluating the determinations. The biuret reaction may also be used to determine the concentrations of pro-
Oct. 1956 PROTEIN ANALYSIS 1173 tein that are obtained by various biochemical procedures, such as the extraction of specific proteins from tissues by means of concentrated solutions of salts or buffers. In such instances, it is suggested that the standard (or standards) and unknown solutions be treated with the same concentrations of salts or buffers in order to obtain valid results. SUMMARY The violet color that is produced by the interaction of copper ions with proteins in alkaline solution {i.e., biuret) is altered by concentrated solutions of inorganic salts. The inclusion of comparable amounts of salts in the standard solutions of protein provides a means of compensating for this "salt effect." Determinations of serum protein by the biuret method, when corrected for the "salt effect," are closely similar to values that are obtained with the classic Kjeldahl procedures. SUMMAKIO IN INTEKLINGUA Le color violette que es producite per le interaction de iones de cupro con proteinas in solution alcalin (i.e., biuret) es alterate per concentrate solutiones de sales inorganic. Le inclusion de comparabiie quantitates de sales in le solutiones standard de proteina provide un medio de compensar iste "effecto de sal." Determinationes de proteina serai per medio del methodo a biuret si adjustate per un tal correction del "effecto de sal" es multo simile al valores obtenite per medio del technicas classic de Kjeldahl. REFERENCES 1. COHN, C, AND WOLFSON, W. Q.: Studies in serum proteins. II. A rapid clinical method for the accurate determination of albumin and globulin in scrum or plasma. J. Lab. & Clin. Med., 33: 367-370, 194S. 2. FISTKR, H. J.: Manual of standardized procedures for spectrophotometric chemistry. Standard Scientific Supply Corporation, Easton, 1950. 3. GOKNAI.L, A. G., BORDAWILL, C. J., AND DAVID, M. M.: Determination of serum proteins by means of the biuret reaction. J. Biol. Chem., 177: 751-766, 1949. 4. HOWE, P. E.: The use of sodium sulfate as the globulin precipitant in the determination of proteins in blood. J. Biol. Chem., 49: 93-107, 1921. 5. KINGSLEY, G. R.: The determination of serum total protein, albumin and globulin by the biuret reaction. J. Biol. Chem., 131: 197-200, 1939. 6. KINGSLEY, G. R.: A rapid method for the separation of serum albumin and globulin. J. Biol. Chem., 133: 731-735, 1940. 7. MAJOOR, C. L. LI.: The possibility of detecting individual proteins in blood serum by differentiation of solubility curves in concentrated sodium sulfate solutions..). Biol. Chem., 169: 5S3-594, 1947. S. MBHL, J. W.: The biuret reaction of proteins in the presence of ethylene glycol..). Biol. Chem., 157: 173-180, 1945. 9. MILNE, J.: Serum protein fractionation: A comparison of sodium sulfate precipitation and electrophoresis. J. Biol. Chem., 169: 595-599, 1947. 10. ROSENTHAL, H. L., AND CUNDIKF, H. I.: A new biuret reagent for the determination of proteins in cerebrospinal fluid. Clin. Chem., in press. 11. WEICIISELBAUM, T. E.: An accurate and rapid method for the determination of proteins in small amounts of blood serum and plasma. Am..). Clin. Path. (Tech. Sect.), 16: 40-40, 1946.