Micromethods for the determination of arginine based on the use of the Sakaguchi reagent have been described (14). This reagent gives a strong color with glycocyamine, arginine, and other monosubstituted guanidine derivatives. 



In a previous communication (5) a method for the determination of glycocyamine was described based on the Sakaguchi reaction and the quantitative separation of glycocyamine from arginine by selective adsorption of the arginine on permutit. In the method outlined below the separated arginine is eluted from the permutit and determined independently

Dubnoff, Jacob W.

English

Caltech Authors - Main

A MICROMETHOD FOR THE DETERMINATION OF 
ARGININE 
BY JACOB W. DUBNOFF 
(From the William G. Rerckho$ Laboratories of the Biological Sciences, 
California Institute of Technology, Pasadena) 
(Received for publication, August 6, 1941) 
Micromethods for the determination of arginine based on the 
use of the Sakaguchi reagent have been described (14). This 
reagent gives a strong color with glycocyamine, arginine, and other 
monosubstituted guanidine derivatives. 
In a previous communication (5) a method for the determination 
of glycocyamine was described based on the Sakaguchi reaction 
and the quantitative separation of glycocyamine from arginine 
by selective adsorption of the arginine on permutit. In the 
method outlined below the separated arginine is eluted from the 
permutit and determined independently. 
A number of common non-chromogenic substances such as 
ammonia, urea, histidine, and creatine reduce the color obtained 
with the Sakaguchi reagent. A change in the usual order of 
addition of the color reagents similar to t,hat previously described 
(5) decreases the interference by these compounds. 
Reagents and Apparatus- 
3 per cent sodium chloride. 
0.3 per cent sodium chloride. 
Permutit according to Folin. Permutit can be regenerated 
after use by allowing 3 per cent sodium chloride to percolate 
through and then washing with distilled water until chloride-free. 
This is most conveniently done in large batches on a Buchner 
funnel. 
10 per cent urea in water. 
0.2 per cent naphthol in absolute alcohol, diluted with 4 volumes 
of the 10 per cent urea solution before use. 
Hypobromite solution. 0.66 ml. of liquid bromine are added 
711 
712 Micromethod for Arginine 
to 100 ml. of 5 per cent sodium hydroxide. Since this solution 
deteriorates, it should not be kept for more than 1 or 2 days, and 
is best kept in a refrigerator. 
10 mg. per cent arginine in 0.1 N hydrochloric acid. Standard 
solutions are made on the day on which they are to be used, by 
diluting this stock solution with water. 
Adsorption column. The permutit is contained in the stem 
of a glass funnel whose dimensions are: upper part 15 mm. external 
diameter, 100 mm. long; stem 7 mm. external diameter, 100 mm. 
long. The lower end of the stem is slightly constricted. A small 
amount of cotton is placed above the constriction. 0.9 gm. of 
permutit is poured in and tapped gently to settle the particles. 
A 0.2 ml. micro pipette whose contents are delivered under 
pressure. The delivery time should be sufficiently short to insure 
rapid delivery and complete mixing (6). 
Preparation of Xolutions for Analysis-For complete separation 
of glycocyamine and arginine the salt concentration of the solution 
should not be over 0.5 per cent. If neither of these compounds is 
present in amounts over 2 mg. per cent, the salt concentration may 
be as high as 1 per cent. Urine is usually diluted 5 to 10 times 
with water. Blood filtrates may be prepared by deproteinizing 
according to Folin and Wu or by heat coagulation at pH 6 after 
1: 10 dilution with water. Tissue extracts are diluted to contain 
1 gm. of tissue (fresh weight) in 40 ml. of suspension. The pH is 
adjusted to 6.0, and the suspension immersed in a boiling water 
bath for 10 minutes, cooled, and filtered. Analyses are carried 
out on the filtrates. 
Procedure 
5 ml. of the solution to be analyzed are passed through the per- 
mutit column and the small amount of glycocyamine remaining 
in the column is removed with 5 ml. of 0.3 per cent sodium chloride. 
The combined filtrate contains all the glycocyamine. A test-tube 
graduated at 10 ml. is now placed under the funnel, and the 
arginine is eluted by passing 10 ml. of 3 per cent sodium chloride 
through the column. The solution in the test-tube is made up to 
the 10 ml. mark and shaken. 
A 2 ml. aliquot is taken for analysis. It is first cooled in an ice 
bath, then 0.5 ml. of the ice-cold naphthol-urea solution is added, 
J. W. Dubnoff 713 
and after 2 minutes 0.2 ml. of ice-cold sodium hypobromite 
solution added by means of the micro pipette. This pipette is 
placed above the solution level and kept away from the sides of the 
test-tube to prevent contamination by urea in the solution. The 
color is simultaneously developed in a series of standard solutions 
containing 0, 0.25, 0.5, 1.0, and 2.0 mg. per cent of arginine.’ 
After 20 minutes the color development is complete and remains 
stable for 2 hours if the solutions are kept in an ice bath. The 
tubes are shaken for a few seconds to remove excess gas, warmed 
by immersion in water at room temperature, and t.he intensity of 
TABLE I 
El&ion of Arginine from Permutit Column 
5 ml. of a 2 mg. per cent solution of arginine in 0.5 per cent sodium 
chloride were passed through the adsorption column followed by 5 ml. of 
0.5 per cent sodium chloride. The arginine was then eluted by the salt 
solution as shown. 
Sodium chloride eluting solution 
Amount Concentration 
ml. 
10 
5 
10 
5 
10 
10 
10 
5 
10 
per oont per cent 
0.5 0 
1.0 2 
1.0 40 
2.0 60 
2.0 100 
3.0 100 
5.0 100 
5.0 58 
10.0 88 
- 
Arginine recovered 
the color measured in a spectrophotometer or a calorimeter, with 
light of approximately 0.525 p (yellow-green). 
DISCUSSION 
As shown in Table I, the complete removal of arginine from the 
permutit column depends on the amount as well as the concentra- 
tion of salt solution used. Under the conditions described, the 
separation of glycocyamine and arginine is complete over a wide 
1 If an appropriate correction factor is applied, glycocyamine standards 
which are stable may be used. 
714 Micromethod for Arginine 
range of concentrations (Table II). With concentrations of 
arginine of over 2 mg. per cent the color is too intense to be read 
and must be diluted before the color is developed. The blanks 
are yellow instead of colorless as in the procedures described in 
the literature. 
In these older procedures a large excess of urea is added after a 
given interval to prevent destruction of the chromogenic compound 
by the hypobromite. During this interval other substances may 
compete for the hypobromite and reduce or completely inhibit the 
formation of color. The advantage of the procedure described 
TABLE II 
Limiting Concentrulions of Glycocyamine and of Arginine That Can Be 
Separated on Permutit Column 
All values are concentrations in mg. per cent. The concentrations were 
those of the solutions passed through the permutit column. The final 
filtrates were diluted to bring the concentration to approximately 1 my. 
per cent before the color was developed. 
I- 
Arginine added Glycooyemine added 
0.0 
0.0 
0.0 
0.0 
1.0 
2.0 
5.0 
10.0 
20.0 
2.0 
2.5 
5.0 
10.0 
0.0 
0.0 
0.0 
0.0 
0.0 
Arginine found 
- 
0.0 
0.0 
0.0 
0.0 
1.0 
2.0 
5.0 
9.6 
18.4 
above is that this interference is greatly reduced and for most 
purposes is negligible. 
Table III shows the recovery of arginine added to urine, kid- 
ney extract, and blood. 
Weber (2) has studied the influence of some non-chromogenic 
substances on color development. He found that 6 mg. per cent of 
ammonia, 12 mg. per cent of histidine dihydrochloride, 6 mg. per 
cent of tyrosine, 6 mg. per cent of tryptophane, 40 mg. per cent of 
creatine, or 160 mg. per cent of urea2 “either prevents all color 
2 These are the concentrations in the final diluted filt,rates. 
J. W. Dubnoff 715 
formation or the color is so altered that even for qualitative pur- 
poses the test is worthless.” With the method described in this 
communication, 60 mg. per cent of ammonia, 5 mg. per cent of 
histidine hydrochloride, 8 mg. per cent of tyrosine, 8 mg. per 
cent of tryptophane, 20 mg. per cent of creatine, and 2000 mg. 
per cent of urea are without influence on the recovery of argin- 
ine. If tryptophane and histidine are present in excess of the 
TABLE III 
Recovery of Arginine Added to Urine, Kidney Extract, and Blood 
All the values are concentrations in the eluate measured in mg. per cent. 
The urine was diluted 1:4 with water. The kidney extract was made by 
grinding up 1 part by weight of kidney with 40 parts of 0.5 per cent sodium 
chloride, bringing the extract to pH 5.0, boiling 10 minutes, cooling, and 
filtering. The analysis was carried out on the protein-free filtrate. In 
both the kidney extract and blood, arginine was added before protein 
precipitation. 
Arginine 
added 
0.0 
0.2 
0.5 
1.0 
1.5 
Found 
0.13 
0.13 
0.345 
0.34 
0.61 
0.61 
1.12 
1.13 
After sub- 
traction of 
,lank value 
0.215 
0.210 
0.48 
0.48 
0.99 
1.00 
Arginine 
Kidney extract T 
Found 
0.15 
0.14 
0.65 
0.655 
1.19 
1.19 
1.64 
1.64 
After sub- 
traction of 
blank vz4ue 
0.505 
0.51 
1.045 
1.045 
1.50 
1.50 
Folin-Wu blood filtrate 
FOUlId 
After sub- 
traction of 
blank value 
0.19 
0.20 
0.41 
0.43 
0.68 
0.68 
1.19 
1.19 
1.67 
1.71 
0.21 
0.22 
0,48 
0.48 
0.99 
0.99 
1.47 
1.51 
amount given, they are chromogenic, and, a further complication, 
histidine reduces the color given by the arginine present. In 
pregnancy urine the concentration of histidine may be as 
high as 160 mg. per cent (7) and the method is inapplicable as it 
stands. The method is satisfactory for normal urine, although 
the possible presence of other chromogens, such as methylguani- 
dine, must be borne in mind. 
716 Micromethod for Arginine 
SUMMARY 
A micromethod for the determination of arginine in biological 
fluids and tissue extracts is described. The advantages of this 
new method are that a complete separation of arginine from 
glycocyamine is effected, and that it gives satisfactory results in 
the presence of common biological substances which interfere in 
the procedure of previous methods. 
BIBLIOGRAPHY 
1. Sakaguchi, S., J. &o&em., Japan, 6, 13, 133 (1926). 
2. Weber, C. J., J. Biol. Chem., 86, 217 (1930); 88, 356 (1930). 
3. Fisher, R. B., and Wilhelmi, A. E., Biochem. J., 32, 606 (1938). 
4. Jorpes, E., and Thoren, S., Biochem. J., 26, 1504 (1932). 
5. Dubnoff, J. W., and Borsook, H., J. Biol. Chem., 138, 381 (1941). 
6. Borsook, H., and Dubnoff, J. W., J. Biol. Chem., 131, 163 (1939). 
7. Racker, E., Biochem. J., 34, 89 (1940). 


A micromethod for the determination of arginine

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A micromethod for the determination of arginine

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