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Verde Janus
Verde Janus
46. MICHAELIS, L., Einfiihrung in die Farbstoffchemie fhr Histologen. S. Karger, Berlin, 1902.
47. NELSON, B. T., Anaf. Record, 23, 355 (1922).
48. PARAT, M., Arch. Anat. Microyop., 24, 73 (1928).
49. QUASTEL,J. H., Biochem. J., 25, 898 (1931).
50. QUASTEL, J. H., and WHEATLEY, A. H. RI., ibid, 25, 629 (1931).
51. RECKNAGEL,R., J. Cellular Comp. Physiol., 35, 111 (1950).
52. HXECHERT,W., and SCHMID, E., Arch. Expft. Path. Pharmakol., 199, 66 (1942).
53. ROBERTSON,T. B., J. Riot. Chem., 2, 317 (1907).
54. RULON, O., Profoptasma, 24, 346 (1935).
55. SCHNEIDER,W. C., J. Riot. Chem., 165, 585 (1946).
56. SCHNEIDER, W. C., and HOGEBOOM,G. H., J. Nail. Cancer Inst., 10, 969 (1950).
57. SEKI, M., Z. Zellforsch. Mikroskop. Anaf., 19, 289 (1933).
58. - ibid, 18, 1 (1933).
59. SHIPLEY, P. G., Anal. Record, 10, 439 (1916).
60. TEAGUE, O., and BUXTON, B., Z. Physik. Chem., 62, 287 (1908).
61. WALKER, A., Proc. Sac. Exptl. Biot. Med., 34, 726 (1936).
T,m J anus Green B sample was purchased from the National Aniline Company.
A concentrated solution was prepared by dissolving 51 mg of the dye in 50 ml of
0.1 M phosphate buffer, pH 7.4. An aliquot removed for nitrogen determination
(by the micro-Kjelda~l method) contained 0.088 mg of nitrogen per ml. Assuming
that all of the nitrogen was from JG-B, the maximum dye content was .534 mg/ml
and the maximum purity of the sample was 52 per cent. A sample of this dye was
also analyzed by the titanous chloride reduction method of Conn (1) and found to
contain approximately 50 per cent JG-B. Assuming a molecular weight of 510 for
JG-B, the molar concentration of this solution was calculated to be about 1.0 x 10-3
M. A dilute solution (1 X 10-3 M) was prepared by diluting with 0.1 M phosphate
buffer pH 7.4. The absorption spectrum was measured in a Beckman spectrophoto-
meter using a cuvette of 1 cm light path.
Attempts to determine the nature of the impurities in the JG-B sample indicated
that it contained 8 per cent water and 11 per cent ash. Thus although the original
sample contained 81 per cent organic material, only 52 per cent is accounted for by
JG-B; therefore approximately 28 per cent of the sample must be non-nitrogenous
organic material. An attempt was made to purify the JG-B sample by recrystalliza-
tion from alcohol at -50 C; however no crystals were formed. The dye was passed
5-533703
70 S. J. Cooperstein, A. Lazarow, and J. W. Pafferson
through two ion-exchange resins, IRC-50 and Dowex-50. The IRC-50 resin did not
adsorb the dye nor did it remove the impurities. The Dowex-50 resin adsorbed the
dye; however 6N HCl was required to remove it from the resin and at this acid con-
centration the JG-B was destroyed.
Janus Green G was kindly supplied by Dr. E. V. Cowdry. A concentrated solution
was prepared by dissolving 48.2 mg in 25 ml of 0.1 M phosphate buffer, pH 7.4. This
solution contained 0.144 mg of nitrogen per ml, and assuming that all of the nitro-
gen was from JG-G the maximum dye content was 0.827 mg/ml. This corresponds
to a purity of 43 per cent. A sample of this dye was also analyzed by the method of
Corm (1) and found to be approxinlately 46 per cent pure. A dilute solution of JG-G
was prepared by diluting the concentrated solution 1: 100 with 0.1 M phosphate
buffer, pH 7.4. The maximum molar concentration of dye in this dilute solution was
1.72 x lo-+ M.
Diethylsafranine. One sample of diethylsafranine was prepared by a modification
of the method of Bensley [quoted by Cowdry (2)]. Sodium hydrosulfite was used as
the reducing agent instead of the zinc and hydrochloric acid recommended by Rens-
ley. The diethylsafranine was precipitated with sodium sulphate and dried. The dye
was extracted with alcohol and the diethylsafranine was obtained by evaporating
the alcohol solution to dryness. If all of the nitrogen in this preparation were from
diethylsafranine, then the sample would be 70 per cent pure, However, since the
molar extinction coefficient at 555 rnp was 20 per cent lower than the molar extinction
of a sample of diethylsafranine prepared by the method described below, it is ap-
parent that dye destruction takes place dnring the above preparative procedure.
The diethylsafranine used for the absorption spectra studies was prepared by
reducing ,JG-B to leucosafranine under controlled conditions. The diethylsafranine
was regenerated by diluting the solution with buffer; further purification was not
attempted. 0.1 ml of 0.1 M sodium hydrosulfite was added to 5 ml of a 1 X lo- M
solution of JGR at pH 7.4. When the resulting leucosafranine was diluted 1 : 10
with phosphate buffer, the dissolved air in the buffer oxidized the excess hydrosulfite
and reoxidized the leucosafranine to diethylsafraninc. This solution was read in the
Beckman spectrophotometer against an appropriate blank, prepared in an identical
fashion except for the omission of the dye. This method of preparation minimizes
the dye destruction since it was found that vigorous shaking of a lcucosafranine
solution, in an attempt to remove the excess hydrosulfite present, resulted in cx-
tensive destruction of the dye.
~~~le~~~~.safFanine was prepared (from JG-G) in the same manner as diethyl-
safranine.
Leucosafranine was prepared by adding 0.1 ml of 0.1 M sodium hydrosulfite to
3.5 ml of a 1.0 x 10-4 M JG-B solution in a Beckman spectrophotometer cuvette.
When the solution became colorless (in a few seconds) a 0.0 mm quartz spacer was
inserted into the cuvette. This reduces the light path of the absorbing solution to
1.0 mm. The tightly fitting quartz spacer minimizes the diffusion of air and thus the
leucosafranine persists for a considerable period. This solution was then read against
an appropriate blank, prepared in an identical fashion except for the omission of the
dye. Although the leucosafranine solution used in the absorption spectra studies
was ten times more concentrated than the other dye solutions used, the light path
was only 1110 as long.
71
YOLECULAR
EXTINCTION
x IO
JANUS GREEN G
I._._._._..
DIETHIL ?A=R&NIHE
LEUCO SAFRLNINE I? !
-..-.**--.*..__ . . ...___.._._. e..
Fig. 1.
*.a -
6.4 -
5P -
5.0 -
..8 -
4.6 -
4.. -
I
!
DIETHYL SAFRANINE.. ,.
.P-
DIMETHYL
.a- /
14- f
3.6 -
3rl- /
32- 1
w- /
:
0.9 - ,J
1.6 -
*.4--
LQ-
eo-
i.* -
1.0 -
1.4 -
I.?. -
LO -
.(I -
.a -
.A--
P-
.Q--
4.0-
31-
6.6 -
Fig. 2.
74 S. J. Cooperstein, A. Lazarow, and J. W. Patterson
Fig. 3.
TABLE I
Calculated Positions of Absorption Spectrum Maxima of Mixtures of Janus Green B and Diethyl-
safranine.
+ IO 20 30 40 90 60 70 Kl 90 I00
I I I I I I I I I
1.6 I
1.7 -
1.6 2
1.2 -
I.1 -
1.0 -
9-
.6 -
.? -
.6 -
.s -
.4 -
3-
.2 -
.I -
0 I I I 1 I / I I
100 90 60 70 60 50 40 30 M IO 0
Fig. 4.
220 260 300 340 360 420 460 500 540 560 620 660 700
Fig. 5.
Janus Green B - mitochondrial staining 77
Fig. 6.
in the extinctions. An inflection appears at about 550 mp. Visually this shift
in position of the absorption spectrum maximum is associated with a color
change from blue (at pH 7.4) to green (at pH 1.4). At strongly alkaline pH
values, there is a decrease in the extinction values.
The absorption spectrum of diethylsafranine shows little significant change
in the pH range of 1.4 to 12.0. The position of the absorption spectrum
maximum does not change and in the pH range between 1.4 and 7.4 the
actual extinction at 555 rnp changes by only 7 per cent.
1 The solutions used in these experiments were partially freed of oxygen by hoiling or by
bubbling helium through them. However no other precautions were taken to maintain anaerobic
conditions.
Janus Green B - mitochondrial staining 79
TABLE II
Chemical Reduction of Janus Green B and G.
The final concentration of the reactants were 1 x 10W5M Janus Green B, 1 x 10e5 M Janus Green G,
.05 M cysteine, .05 M glutathione, and .05 M phosphate buffer, pH 7.4. Each figure represents
the average of 3 experiments.
Janus Green B.
Step 1: Janus green B + diethylsafranine ..... . - .013 - .OOl - .OlO
Step 2: Diethylsafranine -& leuco diethylsafranine .. - .188 - .188 - .217
Step 2: Janus Green G -+? .. .. ....., .. ) -214 /
hoff and Lingane (3).4 This reduction step probably represents the splitting
of the azo bond with the formation of diethylsafranine and p-aminodimeth-
ylaniline. The second reduction step, which occurs at a potential of - .188
to - .217 volts, is probably reversible .4 It presumably represents the con-
version of diethylsafranine to leucosafranine.
We have been unable to detect a polarographic oxidation-reduction step
which would correspond to the reversible reaction JG-B F! leuco-JG-B. It
seems reasonable, therefore, to conclude that this step was either too small
to be detected or that this reaction occurs at a potential so close to the po-
tential at which the azo bond splits that the two steps overlap. Since the
height of the first reduction step (splitting of the azo bond) was much lower
than the height of the second reduction step (reduction of the azine ring) it
is probable that the height of a JG-B s leuco-JG-B step would be much
smaller than the first step and therefore more likely to escape detection.
The polarographic reduction of JG-G at pH 7.48 revealed only one step;
the half-wave potential for this reduction corresponds to the second step
observed in the reduction of JG-B. (See Table III.) The oxidation-reduction
potential of JG-G calculated on the basis of the equation E, =Eo - .0591
pH (3) was found to be - .258 volts.5 This agrees moderately well with the
value of - .285 volts previously obtained (5) by means of an electrometric
titration with TiCl, at pH 7.4.
The absence of an oxidation-reduction step at about - .OOl volts differen-
1 Kindly carried out by Dr. Alan Powell of Case Institute of Technology.
2 0.1 M phosphate buffer.
s 0.1 M borate buffer.
* According to this method one plots the potential at various points in the step against the
logarithm of (I/ICI) where I is the current flowing at the corresponding potential, and Id is the
current flowing at the half-wave potential. If the reduction process is reversible, the resulting
graph is a straight line and the intercept of the plot is equal to the half-wave potential.
6 The use of this equation involves several assumptions which make the oxidation-reduction
potential value so obtained subject to some error.
Janus Green B - mitochondrial staining 81
tiates the reduction of JG-G from that of JG-B. It is possible that this first
step is also present in JG-G but that it is simply too low to be observed. This
is supported by the fact that the height of the second step in JG-G was con-
siderably less than the corresponding step of JG-B. On the other hand the
failure to find the first step in JG-G might also be due to an inherent differ-
ence in the mechanism by which these two dyes are reduced.
DISCUSSION
On comparing the chemical structures of JG-B and JG-G one would hardly
predict that the substitution of two methyl groups for the two ethyl groups
would make any appreciable difference in chemical reactivities of these
compounds. Nevertheless in contrast to the JG-B, JG-G was not appreciably
reduced by .05 M cysteine and it did n.ot have a polarographic reduction
step at the half-wave potential at which the azo bond of JG-B was split.
These results would seem to suggest that the azo bond of JG-G may be re-
duced with greater difficulty than the corresponding linkage in JG-B. How-
ever, strong reducing agents such as hydrosulfite or zinc and hydrochloric
acid will rupture the azo bond of both compounds and produce the corre-
sponding safranine derivatives.
SUMMARY
1. The absorption spectra of JG-B and its reduction products are reported.
JG-B has absorption spectrum maxima at 595, 390, and 285 mp. Diethyl-
safranine has absorption spectrum maxima at 555 and 272 rnp. Leuco-
safranine has no maximum in the visible range.
2. Janus Green G (dimethylsafranineazodimethylaniline) has absorption
spectrum maxima at 587, 395, and 281 mp. Dimethylsafranine has absorp-
tion spectrum maxima at 555 and 275 rnp.
3. Two methods are presented for determining the relative amounts of
JG and the corresponding safranine derivative in mixtures of the two dyes.
4. The effect of pH on the stability of JG-B and its derivatives were studied.
5. Whereas JG-B was readily reduced by cysteine and glutathione, JG-G
was not significantly reduced by cysteine.
6. The polarographic reduction of JG-B and G were studied. At pH 7.48
JG-B showed two reduction steps, one at a potential of - .OOl volts, the
other at - .188 volts. JG-G showed only one reduction step at pH 7.48 (E =
- .214 volts).
REFERENCES
IN this paper we have attempted to delimit the enzyme systems which are
capable of reducing JG-B (to leuco JG-IS, diethylsafranine, and leucosafra-
nine) and to determine whether the components of the cytochrome system
are capable of reoxidizing leuco JG-B and leucosafranine. Earlier work
had suggested that a wide variety of biological agents are capable of reducing
JG-B; indeed the ability of these systems to reduce JG has been used as a
tool for differentiating bacteria (3, 4, 8, 25, 40), as a test for the quality of
milk (17), as a means of determining physiological gradients f 13, 14, 15, IS),
and as a method for studying the oxidation-reduction potential of various
tissues and organisms (1, 2, 38). It has been suggested (7) that this re~luction
process is enzymatic in nature.
In 1933 Banga el al. (5) reported that the lactic dehydrogenase system could
reduce JG-B to diethylsafranine and to leucosafranine. They used an en-
zyme system consisting of washed heart muscle fortified with lactic acid
and coenzyme, and found that this crude preparation reduced JG-B to
diethylsafranine and partially decolorized diethylsafranine under anaerobic
conditions. The first reduction step was found to be irreversible whereas
the second step was reversible. The addition of pyruvic acid inhibited the
decolorization, whereas if the pyruvic acid was added after the leucosafra-
nine was formed the leucobase was partially reoxidized to diethylsafranine.
Apparently an equilibrium state was reached.
1 The authors did not specify which of the various types of Janus Green was used.