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Protein measurement with the folin Phenol Reagent

(From the
Department of Pharmacology,
Washington University
School oj Medicine, St. Louis, Missouri)
(Received for publication, May 28, 1951)
Since 1922 when Wu
proposed the use of the
phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Reagents-Reagent A, 2 per cent N&OX in 0.10
NaOH. Reagent
B, 0.5 per cent CuS04.5Hz0 in 1 per cent sodium or potassium tartrabe.
Reagent C, alkaline copper solution. Mix 50 ml. of Reagent A with 1
ml. of Reagent B. Discard after 1 day. Reagent D, carbonate-copper
solution, is the same as Reagent C except for omission of NaOH. Re-
agent E, diluted Folin reagent. Titrate Folin-Ciocalteu phenol reagent
((II), Eimer and Amend, Fisher Scientific Company, New York) with
NaOH t.o a phenolphthalein end-point. On the basis of this titration
dilute the Folin reagent (about 2-fold) to make it
N in acid. Working
standards may be prepared from human serum diluted IOO- to lOOO-fold
(approximately 700 to 70 y per ml.). These in turn may be checked
against a standard solution of crystalline bovine albumin (Armour and
* Supported in part by a grant from the American Cancer Society on the recom-
mendation of the Committee on Growth of the National Research Council.
This is an Open Access article under the CC BY license.
Company, Chicago); 1 y is the equivalent of 0.97 y of serum protein (see
below). Dilute solutions of bovine albumin have not proved satisfactory
for working standards because of a marked tendency to undergo surface
Procedure for Proteins in Solution or Readily Soluble in Dilute Alkali-
(Directions are given for a final volume of 1.1 to 1.3 ml., but any multiple
or fraction of the volumes given may be employed as desired’.)
To a sample of 5 to 100 y of protein in 0.2 ml. or less in a 3 to 10 ml.
test-tube, 1 ml. of Reagent C is added. Mix well and allow to stand for
10 minutes or longer at room temperature. 0.10 ml. of Reagent E is
added very rapidly and mixed within a second or t,wo (see below). After
30 minutes or longer, the sample is read in a calorimeter or spectrophotome-
ter. For the range 5 to 25 y of protein per ml. of final volume, it is
desirable to make readings at or near X = 750 rnp, the absorption peak.
For stronger solutions, the readings may be kept in a workable range by
reading near X = 500 m/l (Fig. 2). Calculate from a standard curve,
and, if necessary, make appropriate correction for differences between
the color value of the working standard and the particular proteins being
measured (see below).
It is unnecessary to bring all the samples and standards to the same
volume before the addition of the alkaline copper reagent, provided cor-
rections are made for small differences in final volume. The critical vol-
umes are those of the alkaline copper and Folin reagents.
If the protein is present in an already very dilute solution (less than 25
y per ml.), 0.5 ml. may be mixed with 0.5 ml. of an exactly double
strength Reagent C and otherwise treated as above.
Insoluble Proteins, etc.-Many protein precipitates, e.g. tungstate pre-
cipitates, will dissolve readily in the alkaline copper reagent. However,
after proteins have been precipitated with trichloroacetic or perchloric
acid, for example, they will dissolve rather poorly in the 0.1
alkali of
this reagent. They become even harder to dissolve if subsequently ex-
tract.ed with fat solvents, and still more so if dried at 100’.
It is not possible to cover all cases, but the following may be helpful in
measuring the protein of acid precipitates. If the amount of protein is
not great, so that it is spread rather thinly, it will usually dissolve in 3
hour or so in 1
NaOH at room temperature. Therefore, one may add,
for example, 0.1 ml. of 1
NaOH to 5 to 100 y of precipitated protein.
1 For example, with the Klett calorimeter, transfer 25 to 500 y of protein in not
over 1 ml. volume to a calorimeter tube. Add water if necessary to make 1 ml.
Add 5 ml. of Reagent C, and, after 10 minutes, 0.5 ml. of Reagent E. Readings are
taken after 30 minutes with the No. 66 filter. If the readings are too high, substi-
tute the No. 54 filter for sample, standards, and blanks.
After + hour or more, 1 ml. of Reagent D (no NaOH) is added, followed
after 10 minutes by 0.1 ml. of diluted Folin Reagent E as usual.
With larger samples, or very stubborn precipitates, it may be necessary
to heat for 10 minutes or more at 100” in 1
alkali. Although this may
lower the readings, they will be reproducible and can be measured with
similarly treated standards.2
Microanalysis-With a Beckman spectrophotometer adapted to 0.05
ml. volume (12), as little as 0.2 y of protein may be measured with reason-
able precision. Aside from reducing the volumes of sample and reagents,
the only necessary change is to use sufficiently slender tubes for the re-
action. If the tubes are too large in diameter, low values will result.
The following is illustrative of a procedure in which it is desired to pre-
cipitate the protein in order, for example, to measure an acid-soluble con-
stituent of the same specimen. In this example, it is assumed that the
sample volume is negligible. Otherwise a smaller volume of more con-
centrated trichloroacetic acid would be used.
To the sample containing 0.2 to 3 y of protein in a tube of 3 mm. inner
diameter and 4 cm. long,s are added 10 ~1. of 5 per cent trichloroacetic
acid.4 After being mixed and centrifuged, 8 ~1. of the supernatant fluid
are removed. To the precipitate are added 5 p-11. of 8
NaOH. The
sample is thoroughly mixed by tapping or “buzzing,“s and is covered
e Bovine serum albumin is especially difficult to redissolve after precipitation.
Several 40 mg. samples were precipitated with trichloroacetic acid, washed with
alcohol and isopropyl ether, and dried. These samples dissolved very slowly in 2
ml. of 1
NaOH. However, after standing overnight, the protein appeared to be
nearly all dissolved and aliquots gave readings 97 per cent of those obtained with
non-precipitated samples. Other samples were heated for 30 minutes at 100” in 1
NaOH. These samples dissolved and the solution turned slightly yellow. The
final readings checked well but were only 82 per cent of those obtained with non-
precipitated samples. Possibly, heating at lower temperature with the 1
would have sufficed, although heating with weaker alkali would not have been effec-
tive, judging from other experience. The use of stronger alkali than 1
did not
appear to be an improvement.
8 These tubes are cleaned by rinsing with dilute NaOH, boiling in half concen-
trated HNOa, and rinsing several times in redistilled water. Filling or emptying
of a beaker full of tubes (tall form of beaker without lip) is accomplished by slow
centrifugation for a few seconds. For emptying, the tubes are transferred upside
down to a second beaker with a false bottom of stainless steel screen. With the slow
centrifugation required beakers will not be broken.
4 Suitable micro pipettes are the Lang-Levy variety (13, 14). For entering these
narrow tubes the bent tip must be especially short and slender.
5 The tube is held at an angle against a rapidly rotating flattened rod or nail.
Any high speed hand tool mounted in a clamp is satisfactory. The contents of the
tube will mix violently without spilling (15). A similar effect may be had with a
commercial rubber-tipped massage vibrator.
with a rubber cap or Parafilm. After 30 minutes, 50 ~1. of Reagent D
are added and the sample is mixed by “buzzing.” After 10 minutes or
more, 5 ~1. of diluted Folin Reagent E are added with immediate “buz-
zing,” and the samples are read after 30 minutes. Standards are perhaps
best prepared by precipitating 5 ~1. of 5, 10, 20, etc., mg. per cent solu-
tions of serum protein with 5 ~1. of 10 per cent trichloroacetic acid, with
subsequent treatment as for the other samples.
There are two distinct steps which lead to the final color with protein:
(a) reaction with copper in alkali, and (b) reduction of the phos-
phomolybdic-phosphotungstic reagent by the copper-treated protein.
Reaction. wifh Copper in Alkaline Solution-The salient features of this
react.ion follow. (1) The color obtained in the absence of copper is prob-
ably attributable entirely to the tyrosine and tryptophan content (16, 17),
and this is not greatly increased by alkaline pretreatment ((4-6) and Table
I). (2) In the presence of copper, alkaline treatment of proteins results
in a 3- to 15-fold increase in color, but, in contrast, the presence of copper
has only a small effect on the color obtained with free tyrosine and tryp-
tophan (Herriott (17, 18) and Table I). (3) The reaction with copper,
although not instantaneous, is nearly complete in 5 or 10 minutes at room
temperature under the prescribed conditions. Heating to 100” or increas-
ing the concentration of alkali accelerates the reaction with copper without
changing the final color. (4) P re rea ment t t with alkali alone does not
alter the subsequent reaction with copper in alkaline solution. Even pre-
treatment for an hour at 60” with 2
NaOH, or for 5 minutes at 100’
with 1 N NaOH, when followed by the usual copper t.reatment, has almost
no effect on subsequent color. Prolonged heating with strong alkali will,
however, decrease the final color.2
Although the alkaline copper reaction and the biuret reaction appear
to be related, they are not strictly proportional, nor, with different pro-
teins, is the amount of biuret color directly proportional to the increment
caused by copper in the color with the Folin reagent (Table I).
A very small amount of copper is sufhcient to give nearly maximum
final color (Table II). The action does not appear to be catalytic. As-
suming the simple relationship copper + protein ti copper-protein ob-
tains, the data with low copper concentrations may be utilized to calculate
an apparent dissociation constant of 3 X 1OV mole per liter with a maxi-
mum of 1 mole of chromogenic protein-bound copper per 7 or 8 amino
acid residues (Table II).
Mehl, Pacovska, and Winder (19) conclude with Rising and Yang (20)
that in the biuret reaction approximately 1 atom of copper is bound for
Extinction Coejicients
of Proteins
The equivalent extinction coefficient z;~, (or 550)
defined as the optical density
at X = 750 (or 550) rnr with 1 atom of N per liter. Nitrogen was measured by the
Kjeldahl procedure of Miller and Houghton (24). The biurct color was developed
with the reagents of Weichselbaum (25). Source of proteins, crystalline trypsin,
crystalline chymotrypsin, and crystalline bovine albumin, Armour and Company,
Chicago; cytochrome c, Sigma Chemical Company, St. Louis; crystalline zinc in-
sulin, Eli Lilly and Company, Indianapolis; gelatin, Difco Laboratories, Inc., De-
troit; L-tyrosine, Eastman Kodalr Company, Rochester.
Protein Copper absent 1
Alkalinet cop-
per treatment
No pre-
Trypsin................... 733 910
Insulin. 989 998
Chymotrypsin. 278 425
Cytochrome c. . 703 738
Human serum.. . 329 365
Bovine serum albumin. . 312 358
Gelatin. . 79 78
Tyrosine. . . . .I 13,700 13,850
Biuret color
* 30 minutes in 0.1
NaOH at room temperature before addition of Folin
t Regular treatment as described under the procedure.
$ Not valid because of the color of the cytochrome c.
Color Increments from Small
Serum protein concentration, 12.1 y per ml.
and chromogenic-bound copper
(Cu-protein) calculated from
= Cu X protein/Cu-protein = (Cu(tota1 protein -
Cu-protein))/Cu-protein = (Cu(maximum
D. * - ~0. D.))/AO. D. (assuming
that chromogenic copper bound to protein is proportioned to AO. D.).
Total Cu 0. D: at 750 nut AO. D. Ao. D., per cent K Cu-proteint
of maximum A (calculated)
lo-’ Y x IO-’
8 166 88 42 2.9 0.05
20 237 159 76 3.0 0.10
40 267 189 91 2.8 0.12
2000 / 286 208 loo I 0.13
* Optical density.
t Moles per 117 gm. of protein, i.e. per amino acid residue.
each 4 amino acid residues, and Mehl et al. calculated dissociation con-
stants for the reaction copper + protein * copper-protein averaging 10
times larger than the one reported herein for the formation of Folin-reac-
tive material. Thus, of the total possible sites for copper combination,
it would appear that only about half produce significant reduction of the
Folin reagent, and that furthermore this fraction has a considerably greater
affinity for copper than the rest.
0 20 40
80 00 120
FIN. 1. “Persistence of reactivity” was measured by adding Folin reagent to
protein-free alkali; after the given times, copper-treated protein was added in a
small volume, and the color at 750 rnjr was measured 30 minutes later. “Color de-
velopment” refers to a sample of serum protein treated in the regular manner.
The points on “optical density 448 mp” are observed (no protein present); the
curve is theoretical for a monomolecular reaction with a half time of 8 seconds.
Reduction of Folin Reagent-Three main points may be made as follows:
(1) When the Folin reagent is added to the copper-treated protein, maxi-
mum color results if the reduction occurs at about pH 10. (2) At this pH
the reagent is only reactive for a short time (16). It is for this reason
that even a few seconds delay in complete mixing will lessen the amount
of color (Fig. 1). The decrease in reactivity of the reagent appears to be
a function of the disappearance of the original yellow color of the phos-
phomolybdate (half time of 8 seconds (Fig. 1)) and is presumably due to
dissociation of the phosphate from the molybdate. Surprisingly, the color
with protein continues to develop for a number of minutes after the re-
agent itself has become unreactive to freshly added protein (Fig. 1). Pos-
sibly the primary reduction product rearranges, since the absorption spec-
trum changes in shape between 3 minutes and 30 minutes (Fig. 2). (3)
During the 1st minute or so after the addition of the Folin reagent, extra
acid is liberated (Fig. l), which also may result from the dissociation of
the phosphomolybdate. Thergfore, for maximum color, the solution must
I I 1 I I I I
400 500 600
800 900 1000
FIG. 2. Absorption spectra 3 and 30 minutes after the addition of Folin reagent
to a solution containing 23.3 ‘y of serum protein per ml.
I I a I
0 .05 10 .15 .20 .25
FIG. 3. Effect of alkali concentration on final color development. NaOH con-
centration is calculated before addition of 0.1 volume of diluted Folin reagent. Ex-
cept as noted, final concentration of Folin reagent 3 per cent and Na&03 1.6 per
cent. All samples (copper-treated protein) were identical in composition until a
few seconds before addition of Folin reagent (see the text). Final protein concen-
tration 12 y per ml.
be rather well buffered. It was found that a mixture of NaOH, sufficient
to neutralize the excess phosphoric acid, and Na&03, to buffer the mixture
near pH
gives more color than any amount of either reagent alone
(Fig. 3).
Extinction Coeficients and Proportionality-Different pure proteins give
different extinction coefficients with the Folin reagent (Table I). The
extremes were observed with trypsin and gelatin which differed by a factor
of 3 in chromogenicity. It will be seen that without copper much greater
differences occur. The variation in chromogenicity must be kept in mind,
but it is much less marked with mixtures of proteins as found in various
tissues (Table III), and for many purposes is not a serious drawback.
The relation of color to protein concentration is not quite linear (Table
Apparent Protein Content of Whole Tissues (Rabbit) and Tissue Extracts Calculated
from Kjeldahl N and from Folin Color
The tissues were homogenized and precipitated with 5 per cent trichloroacetic
acid (TCA), and the lipides removed by successive extraction with 0.1 N potas-
sium acetate in ethanol, ethanol, and isopropyl ether. (The purpose of the ace-
tate is to neutralize the acid and prevent solution of some protein in the ethanol.)
The N was determined as in Table I. The extinction coefficients were calculated
from the N and color of the extracted precipitates.
Material analyzed
Skeletal muscle
Based on N X 6.25
Folin color
N X 6.25
Folin color
N X 6.25
Folin color
‘I N X 6.25
Folin color
N X 6.25
Folin color
E ktracted TCA
PPt. extract
per cenl
per cent
0.30 0.21
0.49 0.28
0.15 0.09
0.20 0.17
Lipide Whole
rtracted tissue
be* cent
ic, cent
* See Table I.
t By summation; other values are direct determinations.
Specificity and Interfering Substances-Few substances encountered in
biological work cause serious interference. Only a little color was ob-
tained with either acid extracts or the lipides extracted from five different
tissues (Table III). Consequently measurements on non-extracted whole
tissue would be in error by only 3 t,o 6 per cent, whereas values based on N
determination would be overestimated by 15 to 20 per cent.
Uric acid (16), guanine, and xanthine (21, 22) react with the Folin re-
agent. Guanine gives about 50 per cent more color than serum protein,
weight for weight. The color is not enhanced by copper. Curiously, gua-
nosine does not react appreciably. Hypoxanthine gives no color if puri-
fied (21). No more than a trace of color was obtained with adenine, ade-
nosine, cytosine, cytidine, uracil, thymine, or thymidine (see also Funk
and Macallum (22)).
Neither color nor interference with protein color development was ob-
served with the following substances at the given
urea (0.5 per cent), guanidine (0.5 per cent), sodium tungstate (0.5 per
cent), sodium sulfate (1 per cent), sodium nitrate (1 per cent), perchloric
acid (0.5 per cent neutralized), trichloroacetic acid (0.5 per cent neutra-
lized), ethyl alcohol (5 per cent), ether (5 per cent), acetone (0.5 per cent),
zinc sulfate (0.1 per cent), barium hydroxide (0.1 per cent).
Most phenols, except nitrophenols, reduce the reagent (16) ; t,herefore
thymol and to a lesser degree sulfosalicylic acid interfere, whereas picric
oj Small Amounts of Protein from Rabbit
Finn1 volume 0.082 ml.
Optical* den-
sity at 750 mp
1 cm.
at 750 mp
Found Present
0.13 0.16
0.15 0.16
0.14 0.16
0.33 0.33
0.35 0.33
0.34 0.33
0.65 0.66
0.67 0.66
0.67 0.66
ptical’ densit:
at 150 Ill/I
1 cm.
at 750 m&t
Found Present
0.98 1.00
1.03 1.00
0.99 1.00
1.30 1.32
1.31 1.32
1.32 1.32
1.60 1.66
1.62 1.66
1.61 1.66
* Corrected for blank.
acid up to 0.1 per cent is permissible. Glycine (0.5 per cent) decreases
the color with protein by 50 per cent. Hydrazine over 0.5 mg. per cent
increases the blank.
Ammonium sulfate greater than 0.15 per cent final concentration de-
creases color development. This is partly due to a decrease in alkalinity,
and up to 0.25 per cent or so can be tolerated if an equivalent amount
of extra alkali is added to the sample. Extra copper does not seem to
Microanalysis-With final volumes less than 0.1 ml., the amount of
color is proportionately less than on the macro scale, especially if the re-
action is carried out in wide tubes. Extensive testing did not definitely
identify the cause of the decreased color. Neither oxygen, carbon dioxide,
nor glass surface seemed to be involved. The critical step is the period
of standing with alkali and copper.
The practical solution to this interesting difficulty seems to be to use
slender tubes and to run standards under the same conditions. Table IV
illustrates the reproducibility of protein measurements on small brain sam-
ples. Rabbit brain was homogenized and diluted ZOO- to ZOOO-fold. Ali-
quots of 3.6 ~1. were analyzed for protein at a final volume of 0.082 ml.
The amount of protein present was calculated from macro analyses. It
is seen that the error is usually not over 0.02 y.
The measurement of protein with copper and the Folin reagent has cer-
tain advantages. (1) It is as sensitive as with Nessler’s reagent, yet re-
quires no digestion. (2) It is 10 or 20 times more sensitive than measure-
ment of the ultraviolet absorption at X = 280 rnp and is much more specific
and much less liable to disturbance by turbidities. (3) It is several fold
more sensitive than the ninhydrin reaction (23) and is somewhat simpler,
as well as much easier to adapt for small scale analyses. Free amino
acids give much more color than proteins with the ninhydrin reaction,
whereas the reverse is true with the Folin reagent. (4) It is 100 times
more sensitive than the biuret reaction.
There are two major disadvantages of the Folin reaction. (a) The
amount of color varies with different proteins. In this regard it is less
constant than the biuret reaction, but more constant than the absorption
at X = 280 rnp. (b) The color is not strictly proportional to concentration.
From a consideration of the advantages and disadvantages, the reason-
able applications of the copper-Folin reaction would seem to include (1)
measurement of protein during enzyme fractionations, etc., (2) measure-
ment of mixed tissue proteins, particularly when absolute values are not
needed, (3) measurement of very small absolute amounts of protein, or
highly diluted protein (e.g. spinal fluid) or protein mixed with colored
substances or other nitrogen-containing substances, and (4) analyses of
large numbers of similar protein samples, such as antigen-antibody pre-
1. A study is presented of the measurement of proteins with the Folin
phenol reagent after alkaline copper treatment. The basic reactions have
certain peculiarities which need to be taken into consideration in using
this reagent.
2. Directions are given for measurement of proteins in solution and
proteins which have been precipitated with acid, etc. A micro procedure
is also described for the measurement of as little as 0.2 y of protein.
3. The differences in the amount of color obtained with a number of
proteins is recorded. Interfering substances are listed.
4. The advantages of simplicity and sensitivity of this reaction recom-
mend it for a number of biochemical purposes.
1. Wu, H., J. Biol. Chem., 61, 33 (1922).
2. Wu, H., and Ling, S. M., Chinese J. Physiol., 1, 161 (1927).
3. Greenberg, D. M., J. Biol. Chem., 82, 545 (1929).
4. Andersch, M., and Gibson, R. B., J. Lab. and Clin. Med., 18, 816 (1933).
5. Greenberg, D. M., and Mirolubova, T. N., J. Lab. and Clin. Med., 21,431 (1936).
6. Minot, A. S., and Keller, M., J. Lab. and Clin. Med., 21, 743 (1936).
7. Pressman, D., Ind. and Eng. Chem., Anal. Ed., 16, 357 (1943).
8. Heidelberger, M., and MacPherson, C. F. C., Science, 97, 405 (1943).
9. Kabat, E. A., and Mayer, M. M., Experimental immunochemistry, Springfield,
321 (1948).
10. Sutherland, E. W., Cori, C. F., Haynes, R., and Olsen, N. S., J. Biol. Chem.,
180, 825 (1949).
11. Folin, O., and Ciocalteu, V., J. Biol. Chem., 78, 627 (1927).
12. Lowry, 0. H., and Bessey, 0. A., J. Biol. Chem., 183, 633 (1946).
13. Levy, M., Compt.-rend. trav. Lab. Carlsberg, SBrie chim., 21, 101 (1945).
14. Bessey, 0. A., Lowry, 0. H., and Brock, M. J., J. Biol. Chem., 184, 321 (1946).
15. Bessey, 0. A., Lowry, 0. H., Brock, M. J., and Lopez, J. A., J. Biol. Chem., 188,
177 (1946).
16. F&n, O., and Denis, W., J. Biol. Chem., 12, 239 (1912).
17. Herriott, R. M., J. Gen. Physiol., 19, 283 (1935).
18. Herriott, R. M., Proc. Sot. Ezp. Biol. and Med., 48,642 (1941).
19. Mehl, J. W., Pacovska, E., and Winzler, R. J., J. Biol. Chem., 177, 13 (1949).
20. Rising, M. M., and Yang, P. S., J. Biol. Chem., 99, 755 (1932-33).
21. Hitchings, G. I-I., J. BioZ. Chem., 189, 843 (1941).
22. Funk, C., and Macallum, A. B., Biochem. J., 7, 356 (1913).
23. Kunkel, H. G., and Ward, S. M., J. Biol. Chem., 182, 597 (1950).
24. Miller, L., and Houghton, J. A., J. Biol. Chem., 169, 373 (1945).
25. Weichselbaum, T. E., Am. J. Clin. Path., 16, Tech. Sect., 10, 40 (1946).
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Aspergillus niger is widely used as an enzyme source in industries. Considering its enzymic potential, A. niger was studied for its acid phosphatase (EC, orthophosphoric monoester phosphohydrolase), and invertase (EC, β-fructofuranoside fructohydrolase) activity in defined media supplemented with 1%, 3%, or 5% sucrose concentrations. Both these enzymes play a key role in phosphate and carbon metabolism in plants, animals, and microorganisms and hence are interesting from the standpoint of biotechnological applications. Ontogenic changes in extracellular, cytoplasmic, and wall-bound enzyme activities of A. niger were studied. Growth in terms of fresh weight showed inverse correlation with pH. At higher pH values, both enzyme activities were higher in the medium supplemented with low sucrose concentration. It was observed that the more the fresh weight of fungi decreased, the greater was the enzyme activity observed. It is suggested that these enzymes may participate in autolysis of fungi and, on the other hand, could prove to be a potential source of industrial application and exploitation.
... Reduced glutathione (GSH) level was estimated using the protocol reported by Jollow et al [21]. GSH level was presented in μmol GSH/g tissue, while the method described by Lowry et al [22] was used to measure protein concentration in the tissues. ...
... Under these conditions, 1 unit of tyrosinase was defi ned as the amount of enzyme that caused an increase of 0.01 per minute in absorbance at 470 nm. The protein concentration was determined by the Lowry method (Lowry et al. 1951) using bovine serum albumin as the standard. ...
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tyr1, the gene for tyrosinase, which is related to pigmentation of mycelia in dikaryotic strains, has been cloned and characterized from the basidiomycete Polyporus arcularius. The gene was present in a single copy in the genome. The putative amino acid sequence of Tyr1 was very similar to that of gLeTyr from Lentinula edodes. However, the carboxy-terminal region of the amino acid sequence of Tyr1 was variable among L. edodes, Agaricus bisporus, and this fungus. In the 5’-untranslated region near the initiation codon, a consensus sequence to the Dof1 binding site that is involved in light-regulated gene expression in maize was found. Transcription of tyr1 was photoregulated; transcription of tyr1 in P. arcularius was activated in light mycelia and inactivated in the dark mycelia. These results suggest that tyr1 is a light-regulated gene regulated by a Dof-like transcription factor in P. arcularius. Although the enzyme activity was observed only in a dikaryon, tyr1 was transcripted in both dikaryotic and monokaryotic strains. Thus, activation of the precursor of Tyr1 may require a posttranslational processing event that is developmentally regulated.
... Total soluble proteins were determined using the method of Lowry et al. (1951). Solution A, B, C (Lowry solution for protein determination) and folin phenol reagent were prepared and used for protein estimation. ...
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Heavy metals like nickel, cadmium, and chromium found in industrial waste pose a seri- ous threat to living organisms. Nickel is extremely poisonous and causes numerous human ailments when it is exceeded from the permissible limit. The most efficient and cost-effective method of removing nickel from contaminated soil is phytoremedia- tion and chelating agents act as supporting material for phytoremediation. Consequently, the purpose of the current study was to enhance the phytoextraction potential of Brassica napus by using chelating agents such as citric acid and ethylene diamine tetra acetic acid (EDTA). For phytoremediation, experimental treatments were comprised of different levels of citric acid, i.e., 10 mM and 20 mM and EDTA, i.e., 1.5 mM and 2.0 mM and the combinations of both (citric acid + EDTA), i.e., 10 mM + 1.5 mM 20 mM+1.5 mM, 10 mM+2.0 mM, and 20 mM+2.0 mM respectively under Ni toxicity. A control without citric acid and EDTA was kept for comparison. Different growth, physiological, and biochemical attributes were measured and analyzed statistically. Results revealed that the concentration of citric acid (10 mM) and EDTA (1.5 mM) separately and in combination EDTA (1.5 mM)+citric acid (10 mM) performed better for the purpose of phy- toremediation and accelerate phytoextraction of Ni through hyper-accumulated Brassica napus. Highest Ni content (27.33 mg/kg) were observed in plants of Corn-II receiving EDTA followed by CA treatment (24.33 mg/pot) and combined EDTA + CA treatment (24.85 mg/pot). The ability of Rapeseed to bio-accu- mulate heavy metals can be used to reduce the level of contaminants in the soil making it suitable for the cultivation of other metals sensitive food crops. The current work demonstrates the effective application of chelating agents (CA and EDTA) to reduce Ni stress as well as to increase Ni accumulation, a requirement for phytoremediation. It is suggested that B. napus species can be utilized for phytoremediation as it is a good accumulator of Ni and other metals.
... The total proteins in both fractions were estimated by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard for quantification of proteins. ...
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Cytochromes are membrane-bound hemoproteins responsible for the generation of ATP via the electron transport system to fuel the metabolic processes of the organism for their growth. This study reports the properties of cytochromes present in the isolated lichenized fungi of the cultured lichen Usnea ghattensis under optimized conditions. The fungal partner of the cultured lichen Usnea ghattensis contains a, b and c types of cytochromes. The concentrations of a, b and c type cytochromes were found to be significantly high (0.0967, 0.0900, and 0.1030 mM/ mg protein, respectively) in the isolated fungal symbiont of cultured lichen grown in malt-yeast extract medium supplemented with 0.01 mol/l sucrose and 0.01 mol/l polyethylglycol. The results suggest that supplementation of additional carbon sources may play a role in optimizing the growth via activating the cytochrome respiratory system in lichenized fungi.
Effect of different concentrations on total leaf protein content was studied in three economically important plant species, viz., tomato, mung bean and maize. Different treatments of SO2 exposure were administered in artificial fumigation chambers. Maize showed least reduction in total proteins. Tomato exhibited maximum decline in protein content after SO2 fumigation. Relationship between individual and interactive effects of SO2 concentration were analyzed with the help of a statistical regression model. Present study helps to establish a correlation between the total leaf protein content, free radicals, activities of antioxidant enzymes like superoxide dismutases and peroxidases and plant sensitivity to SO2 under ambient conditions as well as in greenhouse environment
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Crystalline 60 per cent active acetyl pepsin has 7 acetyl groups per mol of pepsin, 3 of which are readily hydrolyzed in acid at pH 0.0 or in weak alkali at pH 10.0. The tyrosine-tryptophane content of this acetylated pepsin, measured colorimetrically, is less than pepsin by three tyrosine equivalents. Hydrolysis at pH 0.0 or pH 10.0 of the 3 acetyl groups results in a concomitant increase in the number of tyrosine equivalents. In the pH 0.0 hydrolysis experiment there is also a simultaneous increase in specific activity. The phenol group of glycyl tyrosine is acetylated by ketene under the conditions used in the acetylation of pepsin and the effect of pH on the rate of acetylation is similar in the two cases. It is concluded that the acetyl groups in the 60 per cent active acetyl pepsin, which are responsible for the decrease in specific enzymatic activity, are 3 in number and are attached to 3 tyrosine phenol groups of the pepsin molecule.
A micro-method is described by which as little as 10 gamma of specific precipitate nitrogen may be determined with a fair degree of accuracy. The error in repeated determinations is about +- 2 gamma.
  • H Wu
Wu, H., J. Biol. Chem., 61, 33 (1922).
  • H Wu
  • S M Ling
  • Chinese
Wu, H., and Ling, S. M., Chinese J. Physiol., 1, 161 (1927).
  • D M Greenberg
Greenberg, D. M., J. Biol. Chem., 82, 545 (1929).