reactions of dyes with cell substances · 2003-03-11 · 122 dye reaction on cell substances. ii in...

REACTIONS OF DYES WITH CELL SUBSTANCES

II. THE DIFFERENTIAL STAINING OF NUCLEOPROTEIN AND MUCIN BY THIONINE AND SIMILAR DYES*

BY EDWARD G. KELLEY AND EDGAR G. MILLER, JR.

(From the Department of Biological Chemistry, College of Physicians and Surgeons, Columbia University, New Yorlc)

(Received for publication, February 20, 1935)

Thionine is a metachromatic stain commonly used by histologists. In our preliminary survey of the staining of smears of purified nuclear materials (l), it was noted that thionine gave color variation but not reliable differentiation among the substances. The color variations were not conditioned by pH or by the acidity or basicity of the materials, as with hematoxylin. Thionine has been much used in tissue sections to distinguish empirically between mucin or mucoid (staining violet) and nuclei (blue), and important physiological theories depend on this. We felt that if this metachromatic differentiation could be studied with the purified proteins, the histological test might be validated and information might be gained concerning the mechanism of the differential staining.

The proteins used in our experiments were beef salivary mucin or osseomucoid, and nucleohistone or cr-nucleoprotein. Nucleic acid was also used in some cases. The thionine was the com- mercial product as provided for histological staining; for most of the experiments this was recrystallized from hot water and alcohol.

Thionine is one of a large group of basic dyes which, as Holmes (2) showed, exist in one colored form in concentrated and another

* This work was aided by a grant from The Chemical Foundation, Inc., to this Department.

The data in this paper are taken from a thesis submitted by E. G. Kelley in partial fulfilment of the requirements for the degree of Doctor of Philoso- phy in the Faculty of Pure Science, Columbia University.

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120 Dye Reaction on Cell Substances. II

in dilute aqueous solutions. Dilution definitely changes the one form to the other, a new absorption band appearing toward the red end of the spectrum. At intermediate concentrations both bands are present in varying intensities, depending on the dilution. Holmes (3) suggested that the dichromatism of these dyes was the basis for their histological metachromasy, but reported no experiments on this point.

The differential staining of mucin and nucleoproteins appears to be fundamentally associated with this phenomenon. Many of the various types of basic dyes which show the “dilution shift” were tested and all of them gave the protein differentiation, in each case the color of the mucin stain corresponding to a predomi- nance of the dye form occurring in the concentrated aqueous dye solution (absorbing in the shorter wave-lengths) and nucleoprotein to the dilute dye form (absorbing in the longer wave-lengths).

Thionine has already been mentioned as such a stain. Toluidine blue, familiar to histologists for mucin differentiation, is another. Brilliant cresyl blue gave pronounced color differences with the two proteins, mucin staining an intense violet and nucleohistone a pronounced blue. Neutral red, a weakly basic azine dye which had been briefly investigated in the spectrophotometer and shown to give the shift on dilution, also stained the proteins differentially, mucin giving a bright orange-red, and nucleohistone a deep pur- plish red. Alkylated triaminotriphenylmethane dyes, such as crystal violet and methyl violet, which showed pronounced shifts in their absorption maxima with dilution, were correspondingly strongly metachromatic with the two proteins. Crystal violet stained mucin an intense violet and nucleohistone a bright blue. Methyl violet, a mixture of alkylated rosanilines of lower alkyl content than crystal violet, gave similar differences.

A number of triphenylmethane dyes were tested. Aqueous pararosaniline stained mucin an intense bright red and nucleohistone a deeper red-purple-a definite differential staining, although the color difference is not as great as in the other dyes studied. As Holmes had noted, this dye does not change its absorption peak when its aqueous solution is diluted; but when it is dissolved in alcohol the absorption maximum shifts toward the longer wave-length in a manner similar to, though less than, the effect of alcohol on all of the dyes which do give the dilution shift.


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E. G. Kelley and E. G. Miller, Jr. 121

The differential protein stain colors correspond to the aqueous and alcoholic colors. These facts suggest that alcohol is able to cause the absorption change which water is unable to give in this case, and that the protein metachromasy is again parallel to this same phenomenon.

The other dyes which do not show the change in absorption curves on dilution (such as malachite green, of the diamino- triphenylmethane [series) failed to give detectable color differences between the two proteins.

I= 625 W-l. oJmip.cdI 2= 125 CLM. 1.3m mC@II 3' 25 PT!M. 5~111mCeil 4 =l250 MM. 02mmcdl

01 I I I I I I I I I I I I I I I I I 470 460 490 500 5lO 520 530 540 550 560 570 580 590 600 610 620 630

WAVE LENGTH

FIG. 1. Absorption spectra of aqueous solutions of thionine

Since this dilution shift appears to be fundamentally related to the phenomenon involved in the differential staining as used in histology, we undertook a study of its nature and the factors concerned.

Spectrophotometric curves of thionine at various concentrations from 1250 parts of dye per million parts of water (p.p.m.) to 25 p.p.m. were made. Cells from 0.15 mm. to 5 cm. thick were used. As Beer’s law was not followed, owing to the development of a new band on dilution, the curves were not recalculated to a single scale.


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In a concentrated aqueous solution (1250 p.p.m.) the dye is almost completely in its violet form with maximum absorption at approximately 560 mp, with some evidence of an extension at 600 rnp (Fig. 1). In a dilute solution the dye is almost completely in its blue form with maximum absorption at 600 rnp, and evidence of the presence of some of the violet at 560 mp. At 125 p.p.m. almost equal amounts of both forms are present. Intermediate dilutions show intermediate proportions of the two.

Spectrograph curves of the thionine solutions in theultra-violet showed a narrow absorption band at about 281.2 mp in all concentrations.

14 THIONINE 111 ALCOHOL

12 I- 625 PPM 0.15mm.Cd1 2- 25 ELM. 3.0mm.CelI

IO -i-w-i 08

I.6

06

04 ‘29 02

00 200 250 3w 350 400 WA”E%NGTAoo 550 600 650

FIG. 2. Absorption spectra of alcoholic solutions of thionine

The effect of different solvents was tried. Solution of the dye in 95 per cent ethyl alcohol converted it into its blue form with a peak at 600 rnp, with a very small amount of the violet form indicated by an extension at 560 mp. 625 p.p.m. and 25 p.p.m. gave almost identical curves (Fig. 2). In alcohol the dye followed Beer’s law very closely. Holmes (2) found a similar effect of alcohol with all of the dyes studied. Butyl alcohol gave the same result. Acetone caused a general broadening of the curve, the indi- viduality of the two forms disappearing. The dye was insoluble in ether and chloroform.

Solutions of the dye in acid and base were tested. In the pH range from 1 to 10 (with HCl or NaOH) the curves were identical.


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Above 10, the dye base precipitated. Below -1, it changed to the blue form and a new green form (the dihydrochloride) with a band at about 670 rnp. 10 per cent acetic acid (pH 2.19) converted the dye largely into its blue form (600 mp) with some of the violet (560 mp), although HCI at pH 2.06 had no effect (Fig. 3).

Sodium hydroxide or ammonia caused precipitation of the color base, soluble in butyl alcohol. Its absorption curve was very broad, covering the range of the violet and blue peaks as well as a new peak at about 490 mp.

24

22

2.0

I,8

I.6

FIG. 3. Effect of acid on absorption spectra of thionine

It was thought that the dilution shift might be subject to a specific effect of the anion. The thionine used in all of the above experiments was the dye hydrochloride. The dye acetate and iodide were tested; their solutions were identical in their absorption curves with the dye hydrochloride, and showed the same behavior on dilution.

To test hydration or solvation as the factor responsible for the dilution effect, various amounts of sodium chloride were added to the dye solutions. With 125 p.p.m. of dye, 0.01 to 1 M salt had no effect. With greater salt concentrations, some of the dye was salted out, the supernatant solution showing the proportion of


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colored forms normal for that dilution in pure aqueous solution. Further increases of salt concentration up to the point of complete precipitation of the dye caused a gradual lowering of the entire curve, with no further change in the relative heights of the two peaks. .

To compare changes in ionization of the dye with the dilution effect, the conductivities of various concentrations of the dye were measured. Molar conductance was plotted against the square root of the molar concentration (Fig. 4). On diluting from 1250 to 25 p.p.m. there was an extremely small uniform increase in ionization, insufficient to account for the pronounced color change

FIG. 4. Conductivity of aqueous solution of thionine

over this range. At 25 p.p.m. the curve shows a more rapid rise, increasing greatly in the very dilute solutions. Hydrochlorides of aniline and other amines show a similar effect (4).

During certain stages of mucin-thionine staining at pH values about 9.0 there was evidence of considerable formation of the dye base. As aqueous solutions of thionine did not show dye base at this pH, it was thought that this might be significant regarding the effect of various groups on the surface of the protein. More- over, as Holmes (5) has reported that the dye base of brilliant cresyl blue is formed readily from the dilute form of the dye and not from the concentrated, it was hoped that a study of the base formation might yield some information as to the differences in


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these forms. As the dye base of thionine is insoluble in water and sparingly soluble in organic solvents, brilliant cresyl blue was used.

Absorption curves of brilliant cresyl blue showed the same type of dilution phenomenon as thionine, except that for a corresponding dilution there was not as complete conversion (Fig. 5). The two bands were at 580 mp and 530 rnp. Up to pH 10.5 to 11.0 the addition of sodium hydroxide did not form dye base; the entire absorption curve was lowered with increasing concentration of alkali, but the relative intensities of the two bands remained constant (Fig. 6). At higher pH values with sodium hydroxide the

I6 I I I I I II I I I I I 1

600 620

FIG. 5. Absorption spectra of aqueous solutions of brilliant cresyl blue

curve became very broad and covered the range of the dye base, violet and blue bands. Strong acids caused the formation of a third band and a green solution.

Holmes’ experiments with brilliant cresyl blue and chloroform were repeated and other experiments on the change from blue form to dye base were undertaken.

Solutions of the dye directly in chloroform gave principallythe blue form (Fig. 7). Shaking a water solution of the dye with an equal volume of CC&H caused the formation of the dye base in the chloroform with maximum at 505 rnp (Fig. 8). There was a


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FIG. 6. Effect of alkali on absorption spectrum of brilliant cresyl blue

I I BRILLIhNT CRESYL BLUE

FIG. 7. Absorption spectrum of brilliant cresyl blue in chloroform, and effect of chloroform extraction from water.


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lowering of the pH of the water phase, the decrease depending on the dye concentration, being as much as 0.75 pH unit in the more dilute solutions. Buffering of the water-dye solution prevented dye base formation (Fig. 8). Deutsch (6) and Kolthoff (7) have studied the color changes in indicator pigments at interfaces between water and an immiscible liquid such as benzene or chloroform; the phenomenon seemed to depend on pH changes in the

l-125 IPM.IN WATER TO CHLOROFORM TT7J4y;;;~;;;To

/ 02 - 2/- w WAVE LENGTH 1

460 486 500 520 540 550 560 600 62.0 640 660 680

FIG. 8. Effect of buffer on dye base formation in chloroform extraction of brilliant cresyl blue.

interfaee. It was shown also, with certain dyes, that it was possible to secure the form corresponding to the colored dye base simply by the introduction of a large interface such as that afforded by quartz particles.

It was evident that the blue form of the dye was very readily changed to the dye base, even by the hydroxyl ion concentration of water at an interface between chloroform and water.

Comparison of the formulae of the many dyes listed by Holmes


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(2) reveals a number of points regarding the nature of the dilution shift. Nitrogen certainly appears to be necessary. Apparently

CH, /

this may be present as NHZ, -NHCHs, -N \;

, or even in

CH, a ring (e.g. alizarin blue S-5-R). With thionine there is a shift of approximately +40 mp on dilution. No alkyl groups are present in this case. Introduction of alkyl groups on the nitrogen atoms, as in toluidine blue and methylene blue, increases the magnitude of the shift. With the triaminotriphenylmethane dyes the change does not occur unless alkyl groups are present. Rosaniline and other fuchsins show no dilution effect, but the introduction of alkyl groups causes the shift to take place. The greater the loading of the nitrogen with alkyl groups, the greater is the magnitude of the effect. The positions of the peaks of the concentrated solutions of all of the dyes of this series are almost exactly the same. Phenyl groups attached to the amino nitrogen atoms or to the central nitrogen atoms of the azine, thiazine, or oxazine dyes, greatly repress the amount of shift on dilution; this is illustrated by the phenylated rosanilines and the safranines. The azo dyes show only a small shift on dilution, regardless of the loading on the nitrogen, and none is observed with the diaminotriphenyl- methane dyes.

Holmes also was unable to correlate the phenomenon with changes in dispersion, hydration, or solvation. He concluded that one must assume some type of tautomeric rearrangement, and explained the change in absorption as being due to a change of alkylated amino nitrogen from a trivalent “addition product type” to a pentavalent ammonium salt type. Examination of Holmes’ data does not support this theory. Nor has it been found possible to account for the shift on dilution, common to the many varied dyes, by any type of structural tautomerism that would be appli- cable to all. This same difficulty makes it impossible to correlate these studies with the chain theory of color as used by Watson (8) and others, on the basis of the passage of an electron along a chain of conjugated double bonds.

Moir (9) has proposed a theory of color involving the move- ment of a shared electron around the entire dye molecule from one


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E. G. Kelley and E. G. Miller, Jr.

positive point to another. The time of revolution of the electron in its orbit, when it is excited by light, depends on the type of dye and the volume of the groups. He claimed that changes in the angle of the nitrogen valence bonds affected the molecular volume. He noted that phenazine, phenthiazine, and phenoxazine dyes exhibited two bands in their solutions, and suggested that the positions of the two bands could be used to calculate the different angles of the valency bonds of the nitrogen atom.

It seems possible that application of this theory might lead to a working hypothesis for the dilution phenomenon. The measure- ments which Moir made were on dilute solutions, and it will be remembered that increased number and size of alkyl groups affect the location of the band of the triphenylmethane dyes only in dilute solution and do not affect the location of the band of the concentrated solution. Moir found that increased volume increased the wave-length of the absorbed light. Decreased volume would mean absorption of light of shorter wave-length. Since a nitrogen-containing chromophore group is necessary to show the dilution shift of absorption, it seems possible that a change in the angle of valency bonds of the nitrogen might occur if for some rea- son the auxochrome group were affected by a change in the medium of the dye solution. Such a picture would not be inconsistent with the data on the dilution effect. With dilution or an appro- priate change in solvent the amino nitrogen may be changed from a more compact form to a more extended one.

For further investigation of the staining reactions of mucin and nucleohistone, a somewhat different procedure was employed from that used in the earlier part of the work. Weighed amounts of proteins were triturated with water and transferred to small centri- fuge tubes. They were washed several times and then dye was added to each tube. Uniform times of staining were employed in most cases. The stained proteins were washed free of excess dye (with water or buffer, depending on the staining medium used), pipetted to slides, and examined while wet, then dried at 50”, and examined again. Samples of each dry smear were mounted in balsam; in no case was there any change in color on long standing, and comparison could be made more easily on the transparent balsam mounts.

The effect of the dye concentration was tried. Mucin and


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nucleohistone were stained for 5 minutes in various aqueous concentrations of thionine. The colors of the smears were determined by examination in the color analyzer (10) in terms of wave-length of the visual color. In concentrated dye (1250 p.p.m.) both proteins gave an identical intense violet (433 mu). In 625 and 125 p.p.m., mucin gave the same violet (433 mp); the nucleohistone gave a somewhat more blue color (437.5 and 449 mu). The greatest color distinction between the proteins was seen at 25 p.p.m.; mucin appeared definitely violet (447 mu), while the nucleohistone was a deep blue (470 mp). In 1 p.p.m., mucin was a dull blue- violet (464 mu) and nucleohistone deep blue (476 mu). Osseo- mucoid and a mucin isolated from the Japanese yam, in 25 p.p.m., gave 449 and 452 rnp respectively.

It is evident that both of the proteins will stain predominantly with the violet form in concentrated solutions of the dye where this is present in great excess. On dilution, with formation of the blue-colored dye, the nucleohistone combines with more of this blue form while the mucin stains with the violet form. The concentration of the violet form in the most dilute staining solution was very small and yet the mucin still stained an intense violet.

To test the time factor in the staining, the proteins were placed in an excess of 25 p.p.m. of dye and samples were removed at intervals of 2, 5, 15, 30, and 60 minutes. With increasing time both proteins became more violet. In all cases there was a decided difference in the color of the two proteins, but this distinction was greatest at 5 and 15 minutes, when mucin was an intense violet and nucleohistone a deep blue. At 30 and 60 minutes nucleohistone became more violet, as if the immediate tendency of the protein was to stain with the blue form of the dye and then, after a certain amount of the dye had been taken up, the violet form could exist on the surface of the protein.

Since alcohol had been shown to convert the dye into its blue form, its effect on staining was tried. When the proteins, stained in 25 p.p.m. of aqueous thionine, were washed in alcohol, the mucin smear changed from violet to blue and the nucleohistone became slightly more blue, the resulting color being the same for bot,h. This phenomenon has been noted by Michaelis (11). When the proteins were stained in a 1250 p.p.m. alcohol solution of the dye, both gave an intense blue (the visual wave-length being 476 mp,


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identical with that obtained with nucleohistone in dilute aqueous dye). When water was added to the stained proteins, the mucin became intensely violet; the nucleohistone remained blue. Stained

in 25 p,p.m. of alcoholic thionine, nucleohistone gave an intense blue, and mucin a very faint blue. The alcohol was capable of keeping the dye in its blue form even on the surface of the mucin, where it had previously been converted to the violet form.

pH had a decided effect on the staining of the proteins but not on the color of the stained material. Mucin smears did not stain at any pH below the isoelectric zone; at pH 3 there was very faint

1.6

14

1.2

lx3

0.6

04

l- 125 R?M. pH $8 lVmm&ll 2-Same, Diluted 5 Times IOmm

02 I I I

w WAVELENGTH 460 480 500 520 540 560 580 600 620 640

FIG. 9. Effect of mucin on thionine

violet staining, increasing with rising pH. Nucleohistone stained

blue from pH 1 to 10, the faintest color being in the region of the apparent isoelectric zone (about pH 3). At pH 2 and below the material became more or less sticky and gelatinous and was stained somewhat unevenly as was the case with stained smears of nucleic acid.

As Fig. 9 shows, the mucin in solution at pH 9.8 exerted a very powerful effect on the thionine, the maximum absorption being even more toward the violet end of the spectrum than the position of the maximum of a concentrated aqueous solution; there was


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also a small extension of the curve at the position of the dye base. The blue form of the dye which would be present in equal amount with the violet form at this concentration (125 p.p.m.) in simple aqueous solution was very greatly reduced in amount. This same solution, diluted 5-fold and examined after the pH had again been brought to 9.8, still showed a great proportion of the violet with somewhat less dye base and considerably more of the blue form. An aqueous solution of this degree of dilution, and at any pH up to about 10.5, would have shown a great excess of the blue with only a small amount of the violet form (Fig. 1).

24

2.0

FIG. 10. Effect of nucleohistone and nucleic acid on thionine

Although the nucleohistone was less soluble, it had a pronounced effect. Strongly centrifuged supernatant solutions at pH 9.3 showed a preponderant blue, with a considerable amount of the violet dye (Fig. 10). As with the mucin, the nucleohistone caused a shift in the position of the maximum absorption, in this case toward the red by about 10 rnp. The solution of nucleohistone with the dye at pH 9.3 was not as clear as the mucin solution and a beam of light showed it to be a suspension of ultramicroscopic particles, giving a dichroic effect by reflected and transmitted light.

Since it was possible that the nucleohistone at this high pH


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E. G. Kelley and E. G. Miller, Jr.

might have formed nucleic acid, a number of solutions of nucleic acid and dye were examined. The nucleic acid was compIetely dissolved at pH 9 and showed a strong tendency to keep the thionine in its blue form (Fig. 10). The same shift of the dilute peak toward the red was noted and also an extension to the longer wave side of the position of the concentrated peak. This extension was more pronounced than with nucleohistone. This difference between nucleic acid and nucleohistone was constant under all conditions of staining.

Curves of mucin and nucleohistone with thionine were also made at pH 4.9. The proteins were not dissolved, and were completely centrifuged off. The muein was stained violet and the nucleohistone blue. The supernatant solutions were more dilute, owing to removal of the stain by the proteins, and in each case the proportion of colored forms and positions of the peaks was the same as for plain water solution at this dilution.

Similar curves were obtained with the proteins and brilliant cresyl blue. At the lower pH values (at which the proteins did not dissolve) there was again no other effect than removal of some of the dye with subsequent equilibrium of the dye forms in solution to fit the dilution. At the high pH levels with solution of the protein, there was the same tendency of mucin to form the violet dye (Fig. ll), with a shift of the peak toward the violet end of the spectrum. Unlike thionine, there was very little tendency to form the dye base in these alkaline solutions. Nucleohistone and nucleic acid again caused a preponderance of the blue dye with a shift toward the red (Fig. 12). The same relation of nucleic acid and nucleohistone was evident by the slightly greater proportion of violet dye in the nucleic acid solution. Nucleic acid also dissolved at pH levels as low as 3, and the conversion of the dye to the blue form was the same as at higher levels (Fig. 12).

At the pH of the suspended mucin-dye mixture (4.63) with 125 p.p.m. of dye, the mucin almost completely prevented dye base formation on shaking with chloroform. Curve 1, Fig. 13, on the chloroform phase, shows that some dye base was formed, but comparison of this curve with that of the dye in chloroform from water (Fig. 8) shows that the conversion was slight. The pH of the supernatant solution changed from 4.63 to 4.52, com- pared with the 0.75 pH shift of the water solution of the dye. The


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mucin may have prevented dye base formation by repressing conversion of the violet dye to the blue form from which the base is formed, or simply by its buffering action. At higher pH levels the mucin did not prevent dye base formation (Fig. 13). There

was a shift in pH from 9.37 to 8.30 on shaking the mucin-dye solution with chloroform. With KCl-borate buffer there was no pH shift even with complete dye base formation. Evidently the mucin lacked buffer value at the high pH levels.

BRILLMNTCRfSYL BLUE 04

"' 1160 480 500 520 540 560 580 600 620 640 I30 680

FIG. 13. Effect of mucin on chloroform extraction of brilliant cresyl blue

With nucleohistone and dye solution at pH 5.5, shaken with chloroform, the dye base was formed as readily as from the water solution. At higher pH levels dye base formation was very complete and there was a pH shift of 1.2 units. When a solution of nucleic acid at pH 3 was shaken with chloroform, the dye base formation was completely repressed, and there was no pH change in the supernatant solution. There was certainly considerable attraction of the nucleic acid for the blue dye although probably less than with nucleohistone. The lack of dye base formation


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may have been due to the buffering action of the nucleic acid at this low pH level. At higher pH levels the dye base was formed on shaking the nucleic acid-dye solution with chloroform, and there was a correspondingly large pH change.

Fig. 14 shows the absorption curves obtained from the solid smears of mucin, nucleohistone, and nucleic acid stained in 25 p.p.m. of thionine. As with all of the solid smears from aqueous suspensions, the dispersion was greater and the entire curve was raised. There was definitely a greater proportion of the blue dye in nucleohistone than in nucleic acid smears, as in the case of the

I.0 I.0

08 08

06 06

0.4 0.4 TtilONlNE 25 PPM. 5 MIN. BALSAM MOUN

02 02

0 0 520 520 530 530 540 540 550 550 560 560 570 570 580 580 590 590 Ma Ma 610 610 620 620 630 630 648 648 650 650 E&l E&l 610 610

WAV E LENGTH WAV E LENGTH

FIG. 14. Absorption spectra of thionine-stained tissue substance

solutions. The mucin curve showed a greater proportion of the violet than did nucleohistone and nucleic acid. As with the solutions of the proteins and dyes, the violet peak was located at a shorter wave-length than in water solution, and the blue at a longer.

Holmes and Peterson (12) had noted a displacement of absorption maxima in dyes when passing from solid dye to solution. With the dyes which showed the dilution effect, his curves showed that both of the dye forms were present in the solid smear and in most cases the peaks were displaced. We prepared solid smears of thionine and determined the absorption curve (Fig. 15). Both peaks were present, but the violet peak was shifted to the shorter


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while the blue was shifted to the longer wave-lengths. The violet peak of the solid dye is at the same wave-length as the violet peak in the mucin-thionine solutions. The displacement of the blue peak is less than in the drying of the pure dye; nor does the drying of the thionine-protein combination appear to increase the magnitude of the shift (Figs. 14 and 15).

To determine whether certain factors other than the attachment of the protein with the dye could be responsible for the shift in color, negatively charged colloids (corn-starch and potato starch)

I I I ‘I\-/r I \ I 1

484 500 520 540 560 580 600 6-B 640 ~ 660 680

FIQ. 15. Absorption spectrum of solid thionine

were mixed with thionine and brilliant cresyl blue and examined at pH 4.5 and pH 9. The absorption curves of the starch-dye solutions were the same as those of a water solution of the dye at the same concentration. 1 per cent solutions of pure egg albumin, at pH 4.5 and 9, had no effect. Since the dye base formation with mucin at pH 9.2 might have been due to strongly positive groups (such as guanidine) on the surface of the protein, solutions of guanidine were tested; they caused dye base formation as ammonia and sodium hydroxide had done, but not at pH values below 10.5 to 11. Guanidine chloride buffer at pH 9.3 showed no effect.


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Edestin, containing a high proportion of these groups, stained an intermediate color as if both forms of the dye had been bound.

Thionine or brilliant cresyl blue was added to colloidal silicic acid gels. In most cases a precipitate formed depending upon the pH of the solution and the degree of gelation. The solutions showed a great preponderance of the violet form of the dye (Fig. 16). At pH 9.3 the solution showed excess of violet dye and, as

I I IA I 1

O” 460 480 500 520 540 560 580 600 620 640 660 666

FIG. 16. Effect of silicic acid gel on thionine

with mucin, a small amount of dye base formation. This gives added evidence that the dye base formation on the mucin may have been due to the large interface presented by the colloidal protein with concentration of OH ions in the interface, as in the experiments cited above on the effects of immiscible liquids.

In view of the experimental data described, the histological differentiation of mucin and nucleoprotein by thionine and similar dyes appears to be valid, and to depend on the same phenomenon


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as is involved in the dilution shift of absorption bands. (Under certain conditions, there may be some dye base formation, but this is not part of the fundamental problem.)

The mechanism of this differential staining can be discussed only tentatively. One possibility is that the two proteins have specific chemical affinities for the two forms of the dye. It is difficult to picture any different chemical structures for the two forms, which might be common to the very wide range of molecular dye structures that give the uniform phenomena with the proteins. In discussing the dilution shift of the absorption bands, we have indicated our reasons for believing that it is not based on any tautomerism which might lead to changing affinities. Moreover, under certain conditions the two types of protein show strong staining with both colors. The color can be changed after union with the protein (as when mucin, stained blue in an alcoholic thionine solution, becomes violet when transferred to water). The most consistent interpretation of our data seems to be that when the dye is united to the protein, or at its surface, the form of the dye is determined, rather than that the protein selects one form from the solution.

Another possibility is that the color on the protein is determined by the dye concentration there, just as it is in the aqueous solution. The fact that there can be very faint violet staining of protein (with a small concentration of dye on the protein) and very dense blue staining (with much dye bound) leads us to reject this theory.

A third possibility may be tentatively suggested, based on Moir’s views of color and their theoretical relation to the dilution shift as already discussed. If we suppose that the color change in solution is due to a change in molecular volume of the dye (per- haps because of a change in angle of a nitrogen bond), we imply that, in more firmly binding the dye, the mucin causes a contrac- tion of the molecule into the form of smaller volume so that the light absorbed by the dye is of shorter wave-length. The nucleoprotein, binding the dye less firmly (or at least with less or different electrostatic effect) tends to hold the dye in the form of larger volume, absorbing in the longer wave-lengths. Our data are consistent with the general view that mucin tends to change the dye to the “concentrated” or “contracted” form (as shown by the experiments on staining colors and on the inhibition of dye base forma-


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tion), while nucleoprotein does not tend to “contract” the dye, although it may, under certain conditions, fix the violet form if that form is present in excess.

SUMMARY

All dyes examined, which on dilution show a definite shift of maximum light absorption toward the longer wave-lengths, give a color differentiation between mucins and nucleoproteins parallel to the familiar histological differential by the thionine or toluidine technique: the color of the stained mucin indicates preponderant staining by the dye form occurring in concentrated aqueous solution, while the color taken by the nueleoprotein corresponds to that of the dye in the dilute solution.

This differentiation is not conditioned by the pH, within the limits in which the dye cation can combine with the protein.

Experiments are reported dealing with the nature of the color shift on dilution, and several hypotheses are discussed.

It is postulated that this type of metachromatic histological differentiation of tissue proteins is to be explained on the basis of the same phenomenon that is involved in the dilution shift of absorption maxima. Experiments on the effects of the proteins on the dyes are reported, and possible theories are briefly discussed.

BIBLIOGRAPHY

1. Kelley, E. G., and Miller, E. G., Jr., J. Biol. Chem., 110,113 (1935). 2. Holmes, W. C., Ind. and Eng. Chem., 16,35 (1924). 3. Holmes, W. C., Slain Tech., 1, 116 (1926). 4. Sidgwiek, N. V., and W&don, B. N., J. Chem. Sot., 99, 1118 (1911). 5. Holmes, W. C., J. Am. Chem. Sot., 60,1989 (1928); 62,5305 (1930). 6. Deutsch, D., Ber. them. Ges., 60, 1036 (1927); Kolloid-Z., 43,52 (1927); Z.

physik. Chem., 136, 1353 (1928). 7. Kolthoff, I. M., KoZZoid-Z., 43,51 (1927). 8. Watson, E. R., Color in relation to chemical constitution, New York

(1918). 9. Moir, J., J. Chem. Sot., 119, 1654 (1921); 121, 1555, 1808 (1922); 133,

2792 (1923); 126, 1134, 1548 (1924); 127, 967, 2338 (1925); 129, 1809 (1927).

10. Kelley, E. G., J. BioZ. Chem., 110, 141 (1935). 11. Michaelis, L., Einftihrung in die Farbstoffchemie fur Histologen,

Berlin (1902). 12. Holmes, W. C., and Peterson, A. R., J. Physic. Chem., 34,1248 (1932).


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Edward G. Kelley and Edgar G. Miller, Jr.DYES

MUCIN BY THIONINE AND SIMILAR STAINING OF NUCLEOPROTEIN ANDSUBSTANCES: II. THE DIFFERENTIAL

REACTIONS OF DYES WITH CELL

1935, 110:119-140.J. Biol. Chem.

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