EFFECT OF HIGH SALT CONCENTRATIONS ON COLOR
PRODUCTION OF THE BIURET REACTION FOR
PROTEIN ANALYSIS
HAROLD L. ROSENTHAL, P H . 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 sulfite1 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-workers3 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
E F F E C T OF CONCENTRATION O F N E U T R A L SALT ON PRODUCTION O F C O L O R IN T H E
B I U R E T REACTION*
Saltf
Sodium sulfite
Sodium sulfate
Sodium nitrate
Lithium chloride
Lithium sulfate
Potassium nitrate
Potassium chloride
Sodium chloride
Concentration in the Tube
Change in Color}
millimols
per cent
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
+ 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 t u b e ,
f All of the solutions of salts were adjusted to a p H of 7.0.
} 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
E F F E C T OF CONCENTRATION OF B U F F E R SALT ON PRODUCTION OF COLOR IN T H E
BIURET REACTION*
Buffer Salt
Concentration in the Tube
pH
per cent
millimols
Dipotiissium phosphulc
Disodium phosphate
1.7
1.7
3.4
10.9
10.9
O.S
O.S
1.7
3.4
3.4
Change in Colorf
7.0
11.4
7.0
7.0
11.4
7.0
11.0
7.0
7.0
11.0
-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 t h e appropriate reagent blank.
TABLE 3
COMPARISON OF M E T H O D S FOR T H E D E T E R M I N A T I O N OF S E R U M P R O T E I N
Determination
Total protein
Average
Range
Albumin*
Average
Range
Number of Samples
Kjeldahl Method
Biuret Method
Gm./lOO ml.
Gm./lOO ml.
7.25
5.52 to 11.32
7.29
5.68 to 11.47
3.43
2.31 to 4.39
3.44
2.3S to 4.39
19
14
' D e t e r m i n e d 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 I N T E K L I N G U A
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.
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