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A biuret method for determination of protein in hyamine hydroxide and NCS solutions used for liquid scintillation counting in toluene
ANALYTICAL
BIOCHEMISTBY
A Biuret
Method
25, 406411 (1968)
for Determination
Hydroxide and Scintillation
NCS
M. SCHMUKLER
of Protein
Solutions Counting
in Hyamine
Used for in Toluene
AND M.
Liquid
J. YIENGST
Gerontology Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda ZOOl4, and the Baltimore City Hospitals, Baltimore, Mar&znd %X%24 Received February
21, l%S
An aqueous alkaline protein solution is not miscible with toluene. Therefore, in order to measure the radioactivity of protein samples by liquid scintillation counting in a toluene solution, two nonaqueous solvents have been developed: Hyamine hydroxide (14) and NCF (5). Although Hyamine hydroxide is itself miscible with water, when a Hyamine-protein solution is added to water, a heavy precipitate results. The addition of NCS alone to water produces a heavy precipitate. Because of this, protein determination in Hyamine hydroxide and NCS solutions cannot be accomplished with presently available assays, all of which are performed in an aqueous medium. This paper describes a nonaqueous biuret reaction that permits protein measurement in organic solvent systems, thus allowing radioactivity and protein determinations to be made on aliquots of the same sample. METHODS
Bovine serum albumin and purified rat liver protein were used as protein standards in these experiments. BSA2 was obtained from Sigma Chemical Company. Rat liver protein was prepared as follows: Liver was homogenized in 5% TCA. The pellet was twice extracted for 15 min at 70°C in 5% PCA. It was then washed with 95% ethanol and extracted 5 min at 50” in l/l ethanol/chloroform. The pellet was then washed with l/l acetone/ether and ether, and then vacuum-dried. Hydroxide of Hyamine-10X was obtained from Packard Instrument CO. and NCS from Nuclear-Chicago Corporation. ‘Nuclear-Chicago trademark ‘Bovine serum s,lbumin.
for its quaternary 406
ammonium
base solvent.
NONAQUEOUS
PROTEIN
407
ASSAY
BSA, 10 mg/cc, was dissolved in Hyamine hydroxide and NCS by suspending it, in the solvent, and heating at 50” for 10 min in a water bath. To prepare a Hyamine hydroxide-liver protein standard, 50 mg of protein was suspended in 1.5 cc of water. Then 3.5 cc of Hyamine hydroxide was added. The suspension was mixed vigorously and heated 10 min at 50”. To prepare an NCS-liver protein standard, 60 mg of protein was suspended in 1 cc of water. To this was added 5 cc of NCS with vigorous mixing. The suspension was heated 10 min at, 50°C. To make the biuret reagent,, 225 mg of CuSO,*5H,O was dissoIved in 97 cc of absolute methanol; 1 cc of ethylene glycol was added with mixing followed by 2 cc of tetramethyl ammonium hydroxide (24% solution in methanol, obtained from Matheson, Coleman and Bell). Reagent, was prepared fresh for each set of determinations. The protein sampIe to be assayed was diluted to 1 cc with the appropriate solvent (NCS or Hyamine hydroxide), and 4 cc of biuret reagent added with mixing. The reaction mixture was heated 15 min at 50” in a water bath. Optical density was measured at, 550 mp. .3oor-
;
I
I
I----.
-1
’ -.--/
.25Oc
-I.400
1-i 500
WAVELENGTH FIG. 1. Nonaqueous biuret reaction absorption liver protein dissolved in NCS.
_.._ ~_~ LP~ 600
L--.-J 700
( Mp)
spectrum. SampIe used was purified
RESULTS
Figure 1 shows the absorption spectrum of the nonaqueous biuret reaction. Purified liver protein dissolved in NCS was used as the standard in obtaining these data. Protein dissolved in Hyamine hydroxide gave identical results. Figures 2 and 3 show the linearity of the reaction using BSA in NCS
SCHMTJKLER
AND
YIENGST
,700 I 500
-
,500
-
ci d ,400
-
.300-
I
3 PROT:lN,
2
Oh
W&T
7
Mg. FIG.
.Boo--~--
2. Standard
T-
T-----
curve.
T---
BSA
in NCS.
-- r-m-1
.700-
.600
-
500d cj
,400
.300-
,200
FIG. 3. Standard
curve,
BSA
in Eyamine
hydroxide.
0
NONAQUEOUS
PROTEIN
409
ASSAY
.a008 .700
-
.600
-
.500ci 0
.400-
.300-
.200-
.IOO-
FIG.
4. Standard
curve,
liver
protein in
NCS.
and Hyamine hydroxide. Figures 4 and 5 show the same with liver protein as the standard. Table 1 compares the chromogenicities of equivalent amounts of purified protein from varying sources. Heart, skeletal muscle, brain, and kidney protein were isolated from rat organs by the same procedure used for liver. Quantity of protein was determined by the micro-Kjeldahl procedure (6) as well as by dry weight. Note that equivalent quantities of different proteins differ considerably in their degree of color production. TABLE 1 of Protein Source on Color Production by the Nonaqueous Biuret Reaction as described in the text; samples dissolved in Hyamine
Influence (protein
sources
BSA Heart Skeletal Brain Liver Kidney
muscle
hydroxide)
AOD/mg protein (dry weight)
AOD/mg protein (Kjeldahl)
0.0825 0.093 0.089 0.082 0.078 0.080
0 ,086 0.102
0.097 0.088 0.0875 0.0937
410
SCHMUKLER
.600
AND
YIENGST
-
Mg. PROTEIN,
DRY WEIGHT
Fro. 5. Standardcurve, liver protein in Hyamine hydroxide. DISCUSSION Several substitutions distinguish this method from the standard biuret reaction. Tetramethyl ammonium hydroxide was found to be a satisfactory replacement for sodium hydroxide. Since sodium potassium tartrate was not soluble in sufficient quantity in methanol, the free acid was tried in place of the salt. Although the free acid was readily soluble, maximum color could not be obtained within a reasonable period of time when it was used. Ethylene glycol was employed instead to stabilize the copper complex. This was suggested by its previous use in the aqueous biuret procedure (7). Initially the reaction was run at room temperature, and optical density was measured 1 hr after addition of the biuret reagent. The standard curves were linear but measurements made at 2, 3, and 4 hr showed that the optical density increased approximately 4% per hour. Heating for 15 min at 50” immediately after addition of the biuret reagent hastened the completion of the reaction, producing a color which was stable for at least 2 hr at room temperature.
Standard curves are linear from 0.5 to 8 mg protein (dry weight) in 5 cc of reaction mixture. The nonaqueous biuret reaction gives OD values about 30% higher than does the standard procedure.
NONAQUEOUS
PROTEIN
Protein standards dissolved in Hyamine several weeks at -20°C.
411
ASSAY
hydroxide
or NCS are stable
SUMMARY
A procedure for measurement of protein concentration in Hyamine hydroxide or NCS solution is described. The assay is linear from 0.5 to 8.0 mg. Use of this procedure offers the advantage of allowing liquid scintillation counting in toluene and protein assay on aliquots of the same sample. REFERENCES 1. VAUQHAN, 2. STEINBERG,
“Liquid
3. 4.
5. 6. 7.
M., STEINBERG, D., AND Lo12.4~. J., Science 126, 446 (1957). D.,
VAUGHAN,
M.,
ANFISSIW,
C.
B.,
GORRY,
J.
D.,
AND
LOGAN,
Scintillation Counting,” p. 230. Pergamon, New York, 1958. HEBBERC, R. J., Science 128, 199 (1958). HERBERG, R. J., Anal. Chem. 32, 42 (1960). HANSEN, D. L., AND BUSH, E. T., Anal. Biochem. 18, 320 (1967). HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D., J. Biol. Chem. 176, 1401 (1948). MEHL, J. W., J. BioF. Chem. 157, 173 (1945).
J.,
BIOCHEMISTBY
A Biuret
Method
25, 406411 (1968)
for Determination
Hydroxide and Scintillation
NCS
M. SCHMUKLER
of Protein
Solutions Counting
in Hyamine
Used for in Toluene
AND M.
Liquid
J. YIENGST
Gerontology Research Center, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda ZOOl4, and the Baltimore City Hospitals, Baltimore, Mar&znd %X%24 Received February
21, l%S
An aqueous alkaline protein solution is not miscible with toluene. Therefore, in order to measure the radioactivity of protein samples by liquid scintillation counting in a toluene solution, two nonaqueous solvents have been developed: Hyamine hydroxide (14) and NCF (5). Although Hyamine hydroxide is itself miscible with water, when a Hyamine-protein solution is added to water, a heavy precipitate results. The addition of NCS alone to water produces a heavy precipitate. Because of this, protein determination in Hyamine hydroxide and NCS solutions cannot be accomplished with presently available assays, all of which are performed in an aqueous medium. This paper describes a nonaqueous biuret reaction that permits protein measurement in organic solvent systems, thus allowing radioactivity and protein determinations to be made on aliquots of the same sample. METHODS
Bovine serum albumin and purified rat liver protein were used as protein standards in these experiments. BSA2 was obtained from Sigma Chemical Company. Rat liver protein was prepared as follows: Liver was homogenized in 5% TCA. The pellet was twice extracted for 15 min at 70°C in 5% PCA. It was then washed with 95% ethanol and extracted 5 min at 50” in l/l ethanol/chloroform. The pellet was then washed with l/l acetone/ether and ether, and then vacuum-dried. Hydroxide of Hyamine-10X was obtained from Packard Instrument CO. and NCS from Nuclear-Chicago Corporation. ‘Nuclear-Chicago trademark ‘Bovine serum s,lbumin.
for its quaternary 406
ammonium
base solvent.
NONAQUEOUS
PROTEIN
407
ASSAY
BSA, 10 mg/cc, was dissolved in Hyamine hydroxide and NCS by suspending it, in the solvent, and heating at 50” for 10 min in a water bath. To prepare a Hyamine hydroxide-liver protein standard, 50 mg of protein was suspended in 1.5 cc of water. Then 3.5 cc of Hyamine hydroxide was added. The suspension was mixed vigorously and heated 10 min at 50”. To prepare an NCS-liver protein standard, 60 mg of protein was suspended in 1 cc of water. To this was added 5 cc of NCS with vigorous mixing. The suspension was heated 10 min at, 50°C. To make the biuret reagent,, 225 mg of CuSO,*5H,O was dissoIved in 97 cc of absolute methanol; 1 cc of ethylene glycol was added with mixing followed by 2 cc of tetramethyl ammonium hydroxide (24% solution in methanol, obtained from Matheson, Coleman and Bell). Reagent, was prepared fresh for each set of determinations. The protein sampIe to be assayed was diluted to 1 cc with the appropriate solvent (NCS or Hyamine hydroxide), and 4 cc of biuret reagent added with mixing. The reaction mixture was heated 15 min at 50” in a water bath. Optical density was measured at, 550 mp. .3oor-
;
I
I
I----.
-1
’ -.--/
.25Oc
-I.400
1-i 500
WAVELENGTH FIG. 1. Nonaqueous biuret reaction absorption liver protein dissolved in NCS.
_.._ ~_~ LP~ 600
L--.-J 700
( Mp)
spectrum. SampIe used was purified
RESULTS
Figure 1 shows the absorption spectrum of the nonaqueous biuret reaction. Purified liver protein dissolved in NCS was used as the standard in obtaining these data. Protein dissolved in Hyamine hydroxide gave identical results. Figures 2 and 3 show the linearity of the reaction using BSA in NCS
SCHMTJKLER
AND
YIENGST
,700 I 500
-
,500
-
ci d ,400
-
.300-
I
3 PROT:lN,
2
Oh
W&T
7
Mg. FIG.
.Boo--~--
2. Standard
T-
T-----
curve.
T---
BSA
in NCS.
-- r-m-1
.700-
.600
-
500d cj
,400
.300-
,200
FIG. 3. Standard
curve,
BSA
in Eyamine
hydroxide.
0
NONAQUEOUS
PROTEIN
409
ASSAY
.a008 .700
-
.600
-
.500ci 0
.400-
.300-
.200-
.IOO-
FIG.
4. Standard
curve,
liver
protein in
NCS.
and Hyamine hydroxide. Figures 4 and 5 show the same with liver protein as the standard. Table 1 compares the chromogenicities of equivalent amounts of purified protein from varying sources. Heart, skeletal muscle, brain, and kidney protein were isolated from rat organs by the same procedure used for liver. Quantity of protein was determined by the micro-Kjeldahl procedure (6) as well as by dry weight. Note that equivalent quantities of different proteins differ considerably in their degree of color production. TABLE 1 of Protein Source on Color Production by the Nonaqueous Biuret Reaction as described in the text; samples dissolved in Hyamine
Influence (protein
sources
BSA Heart Skeletal Brain Liver Kidney
muscle
hydroxide)
AOD/mg protein (dry weight)
AOD/mg protein (Kjeldahl)
0.0825 0.093 0.089 0.082 0.078 0.080
0 ,086 0.102
0.097 0.088 0.0875 0.0937
410
SCHMUKLER
.600
AND
YIENGST
-
Mg. PROTEIN,
DRY WEIGHT
Fro. 5. Standardcurve, liver protein in Hyamine hydroxide. DISCUSSION Several substitutions distinguish this method from the standard biuret reaction. Tetramethyl ammonium hydroxide was found to be a satisfactory replacement for sodium hydroxide. Since sodium potassium tartrate was not soluble in sufficient quantity in methanol, the free acid was tried in place of the salt. Although the free acid was readily soluble, maximum color could not be obtained within a reasonable period of time when it was used. Ethylene glycol was employed instead to stabilize the copper complex. This was suggested by its previous use in the aqueous biuret procedure (7). Initially the reaction was run at room temperature, and optical density was measured 1 hr after addition of the biuret reagent. The standard curves were linear but measurements made at 2, 3, and 4 hr showed that the optical density increased approximately 4% per hour. Heating for 15 min at 50” immediately after addition of the biuret reagent hastened the completion of the reaction, producing a color which was stable for at least 2 hr at room temperature.
Standard curves are linear from 0.5 to 8 mg protein (dry weight) in 5 cc of reaction mixture. The nonaqueous biuret reaction gives OD values about 30% higher than does the standard procedure.
NONAQUEOUS
PROTEIN
Protein standards dissolved in Hyamine several weeks at -20°C.
411
ASSAY
hydroxide
or NCS are stable
SUMMARY
A procedure for measurement of protein concentration in Hyamine hydroxide or NCS solution is described. The assay is linear from 0.5 to 8.0 mg. Use of this procedure offers the advantage of allowing liquid scintillation counting in toluene and protein assay on aliquots of the same sample. REFERENCES 1. VAUQHAN, 2. STEINBERG,
“Liquid
3. 4.
5. 6. 7.
M., STEINBERG, D., AND Lo12.4~. J., Science 126, 446 (1957). D.,
VAUGHAN,
M.,
ANFISSIW,
C.
B.,
GORRY,
J.
D.,
AND
LOGAN,
Scintillation Counting,” p. 230. Pergamon, New York, 1958. HEBBERC, R. J., Science 128, 199 (1958). HERBERG, R. J., Anal. Chem. 32, 42 (1960). HANSEN, D. L., AND BUSH, E. T., Anal. Biochem. 18, 320 (1967). HILLER, A., PLAZIN, J., AND VAN SLYKE, D. D., J. Biol. Chem. 176, 1401 (1948). MEHL, J. W., J. BioF. Chem. 157, 173 (1945).
J.,