1. Freeze Drying
Freeze-drying is the most desirable method for the preparation of the specimens, both on the theoretical grounds mentioned later and on the empirical ground that a high and reproducible level of reaction is thus attained.
For tissue sections, standard methods of freeze-drying (cf., e.g. Bell, 1956) small pieces of tissue are satisfactory. The problem of freeze-drying thin smears will receive attention here, since (i) some specimens, e.g. ascites tumour, must be examined thus, and (ii) more importantly, normal tissues can often with advantage be examined in smear form for exact micro-spectrophotometry. A small fragment of the tissue (e.g. liver, kidney, etc.) is tapped with a perfectly flat-ended rod in a little isotonic saline for disruption, and smeared on a cover-slip, which is immediately quenched in a stirred bath of ^o-pentane : propane (1:2) at liquid nitro
gen temperature. The pressure exerted during the tapping and the smearing determines whether nuclei are liberated, and to what extent, from the separated cells. The saline can be replaced by other media, e.g.
Tyrode solution, sucrose solutions, etc., depending on the degree of retention required. No medium at all need be used, although free tissue fluids are always present ; a smearing-squashing technique is then still
A C Y L A T I O N A N D D I A Z O N I U M C O U P L I N G 227 satisfactory for most tissues, but some thick regions may be present and must be ignored. I n the BDC reaction, the intensity has been found in practice to be the same whether the nuclei are in intact cells, or liberated in saline or without external medium. A similar smearing technique has been used by Β. M. Richards in Feulgen measurements of the DNA content per nucleus (cf. Richards et al., 1956).
The quenched smears are washed by dipping in liquid nitrogen and are rapidly transferred to the drying chamber (see Appendix 3). Drying is carried out at —50°, although any temperature below about —40° is permissible, the rate of drying decreasing with decrease in temperature.
I t is essential to maintain the smear surface at a fairly uniform tempera
ture. Unevenly dried or poorly preserved specimens give a decreased or variable reaction. When dry, the smears are raised to room temperature in vacuo, and rapidly removed to a desiccator for storage.
The reaction cannot be obtained on smears or sections prepared by the method of freezing-substitution (Simpson, 1941) using absolute methanol or ethanol.
2. Specimen Pre-treatment
Smears for micro-spectrophotometry are simply fixed in absolute ethanol immediately prior to reaction. Frozen-dried tissue blocks are embedded in wax in vacuo and sectioned. Mounting on slides must be performed either by slight warming alone, or by flattening on acetonitrile or on 95% alcohol (e.g. at 47°, 30 sec). The water present in the latter medium does not appear to affect the wax-infiltrated tissue in these conditions. The wax is removed by xylene when required.
Fixation methods other than freeze-drying have not been studied in detail on account of their uncertain effects on the nucleoprotein. Tissue fixed in bulk in absolute alcohol, or in Lewitsky's fluid, gives the reaction, but to what quantitative extent is undetermined. After some fixatives, e.g. Carnoy's fluid, the reaction is weak or variable.
3. Reaction Method
(i) Fixation in absolute alcohol, two baths . . 20 min (ii) Wash in dry acetonitrile, two baths . . . 6 min (iii) Benzoylation: Dry acetonitrile (50 ml), benzoyl
chloride (4-2 ml) and dry pyridine (2-2 ml). Used at room
temperature, in a desiccator (CaCl2) . . . . 3 hr (iv) Alcohol wash, two baths . . . 6 min
(v) Take down to water through 90, 70, 50 and 25%
alcohols.
228 Ε. Α. BARNARD
(vi) Coupling (all operations in this stage are performed in an ice bath: specimens must be at 2°-4° throughout).
Wash in ice cold water . . . 4 min Tetrazotized dianisidine: a solution (0-04%) of the
stabilized salt (Appendix 2) in sodium veronal (2%) buffer, p H 9-0. This solution is made immediately before use and filtered quickly in the cold through a coarse paper. It is a clear yellow solution, which darkens on standing. Normal exposure is 18 min.
Washes: Veronal buffer (0-2%, p H 9) . . 1 min Water, two baths . . . . 1 min each
0-05 N HC1 l m i n Water, two baths \ min and 1 min
Second coupling: ΙΪ acid ( 0 - 1 % solution of the Na^salt)
in carbonate-bicarbonate buffer, p H 9 . . . . 1 0 min Or β-naphthol (0-1%) in N a2C 03 solution (0· 5%) . 10 min The specimens are then allowed to warm to room temperature, in the second coupling bath, over a period of 20 min.
(vii) Wash in N a2C 03 solution (0*5%), two baths . 6 min Wash in water, two baths . . . . 6 min (viii) Dehydrate through 25, 50, 70 and 90% alcohols.
Absolute alcohol, two baths . . . 6 min
Alcohol-xylene (1:1) l m i n
Xylene, two baths . . . 6 min
(ix) Mount in balsam, or for measurement mount and store in Shillaber's immersion oil under cellophane.
Some of these periods are flexible (see B, 3 below). Precautions must be taken to ensure that no moisture comes into contact with the specimen from the stage of freeze-drying to the end of benzoylation.
4. Micro-spectrophotometric Method
Measurements on this reaction have so far been carried out using the scanning, integrating micro-spectrophotometer of Deeley (1955).
The procedure used, described below, is similar to that of Deeley etal., (1954) and of Richards et al., (1956) for measurements of Feulgen stain.
Other micro-spectrophotometric methods, of proven reliability else
where, can no doubt be applied here.
The specimen (here, in all cases, a smear on a cover-slip) is taken from the temporary mounting in immersion oil, washed in xylene and alcohol and in 90, 70, 50 and 25% alcohols and water, and drained. I t is mounted
ACYLATION AND DIAZONIUM COUPLING 229 in glycerol under cellophane, and secured to a brass holder by wax.
Objective and condenser lenses are immersed in Shillaber's oil. Nuclei are crushed by the crushing condenser, then totally enclosed by the dia
phragm, and measured. The reading, at the same diaphragm setting, for an adjacent blank area is subtracted, to give the integrated total absorp
tion (in arbitrary units) for that nucleus. The mean of three readings is taken in every case.
Nuclei are measured in a number of different areas selected at random on the same specimen. Where significant variability among similar cells is found between different regions of one specimen, it is discarded : this can occur if drying was not satisfactory and is usually correlated with inferior cytological preservation.
B . E F F E C T OF VARIABLES IN THE CYTOCHEMICAL PROCEDURE
In establishing the conditions giving the most satisfactory and reproducible results with the maximum production of colour, frozen-dried rat liver and kidney, and chicken or frog blood smears have been used, in both qualitative and quantitative observations.
1. Coupling
Reaction using o-dianisidine gives, as expected, a rather stronger colour than using benzidine. p H around 9 seems optimum; adsorption of decomposition products becomes appreciable at higher pH, necessitat
ing further washing with the risk of some loss of the free diazo group.
70 60 50
Mean stai
n 40
30 20 10
0 10 20 30 40 50 60
Exposure to TDA (min.)
F I G . 6 . V a r i a t i o n of B D C s t a i n i n g w i t h l e n g t h of e x p o s u r e t o s t a n d a r d t e t r a z o s o l u t i o n ( c o n c e n t r a t i o n of free t e t r a z o n i u m d i c h l o r i d e , 0 - 0 1 7 % ) . F r o g e r y t h r o c y t e s ; e a c h p o i n t r e p r e s e n t s t h e m e a n s t a i n ( + S.E.), i n a r b i t r a r y u n i t s , for a b o u t 3 0 nuclei.
230 Ε . Α . B A R N A R D
Concentrations of (free) tetrazotized dianisidine much above 0-02% also incur this danger. The wash with dilute HC1 is intended to destroy the acid-labile triazenes (cf. Section I I , A). The concentration used, 0-05 N, is that which was shown, in work on tissue in bulk, to be the lowest that would give maximum splitting at those sites.
With other conditions standardized, the extent of reaction has been measured, in the nuclei of frog red cells, as a function of the exposure to the tetrazonium reagent (Fig. 6). The stain increases with length of reaction time to reach a plateau of maximum stain. At 60 min, however, there are signs that decomposition products are accumulating. Fourteen to 30 min is optimal.
Of the naphthols tried (Table I), H acid gives a reaction product with the most suitable light absorption properties for micro-spectrophoto
metry of the present type. In measurements oji frog red cells, it has been found that the intensity is unchanged for exposures to H acid solution, from 4 min to 12 min at 2°, followed by warming up to room temperature,
the total period in H acid being constant at 30 min. Coupling in H acid immediately at room temperature, however, gives a decrease (15%) in mean intensity.
2. Benzoylation
Qualitatively, the same pattern of dependence on length of benzoyl
ation has been observed on a number of different tissues, namely the cytoplasmic stain (apart from the special cases described in Section VI, B) decreases rapidly over the first 30 min and appears very slight or neglig
ible at 1^ hr and nil thereafter. The nuclear stain persists after at least 20 hr benzoylation. The concentration of benzoyl chloride can be reduced to as low as 1*5%: 18 hr treatment then gives the same result as 10%
benzoyl chloride for 3 hr.
Quantitatively, the conclusions with regard to time dependence have been confirmed in the case of frog red cells (Fig. 7). I t is seen that from 2 to 20 hr the nuclear stain remains constant. Cytoplasmic stain is zero at
2 hr. Hence the difference in reactivity involved is not merely kinetic, but is so great as qualitatively to distinguish this nuclear component.
Measurements after less than 2 hr benzoylation are complicated by the cytoplasmic stain remaining, i.e. the nuclear stain cannot be measured in whole cells by the present method without the inclusion of stain (where present) in some adjacent or overlying cytoplasm. I t might be of value to measure nuclei isolated by the non-aqueous method (Allfrey et al., 1952) to examine the initial total nuclear stain and its change with benzoyla
tion. Routinely, 3 hr benzoylation is satisfactory. Exposures as long as 20 hr give detectable swelling of some structures.
A C Y L A T I O N A N D D I A Z O N I U M C O U P L I N G 231 Benzoyl chloride (10%) in dry pyridine alone gives some blocking, but the medium develops a strong red-purple colour which tends to stain parts of some tissues. This colour is similarly obtained with rigorously purified pyridine, and is due to an undesirable secondary reaction. This is reduced to a low level in the acetonitrile medium. Benzene and similar vehicles have also been tried, but a precipitate of the quaternary benzoyl salt of pyridine is present in these. The omission of a tertiary base gives,
60 h
40F
20 h
12 (hr.)
16 20 Benzoylation
F I G . 7. V a r i a t i o n of B D C s t a i n i n g w i t h l e n g t h of b e n z o y l a t i o n p e r i o d . F r o g e r y t h r o c y t e s ; e a c h p o i n t r e p r e s e n t s t h e m e a n s t a i n ( + S.E.), in a r b i t r a r y u n i t s , for a b o u t 30 nuclei. T h e o p e n circle d e n o t e s a p p a r e n t h i g h e r s t a i n w h e n c y t o p l a s m i c b l o c k i n g is i n c o m p l e t e ( b u t t h e m e a s u r e m e n t s a r e , for t h a t r e a s o n , less reliable a t t h a t s t a g e ) . ( F r o m B a r n a r d , 1960a.)
as expected, slow and inefficient benzoylation. Trimethylamine can replace pyridine, but secondary reactions, forming a colour in the medium, are more pronounced with this base.
Controls, subjected to the acetonitrile-pyridine solution with benzoyl chloride omitted, have been applied in all tissues examined, and give a subsequent coupling reaction identical with that obtained in an untreated parallel specimen.
3. Other Variables
Micro-spectrophotometry of frog red cell and mouse kidney smears has shown that no detectable change in the amount of the BDC stain occurs with (a) extension of the initial alcohol fixation up to at least 24 hr ; (b) length of time in alcohol after benzoylation up to at least 20 hr;
(c) length of time in water before coupling, up to at least 2 hr ; (d) length of time in alcohol after coupling, up to at least 3 hr ; (e) length of storage in Shillaber's oil, from nil to at least 1 month (at 0°C).
232 Ε. Α. BARNARD
G. PROCEDURES FOR THE ESTABLISHMENT OF THE CHEMICAL BASIS OF THE CYTOCHEMICAL REACTION
The methods described in this section have had as their object the reaction of nuclei in bulk, followed by fractionation, separation of a fraction containing the introduced dye groups, and analysis to identify the sites of reaction. Such methods should be applicable, with appropriate modifications, to the analysis of other reactions of this type (cf., for example, Maddy, 1961). Chicken erythrocyte nuclei were chosen as material in the present case, on account of their ease of preparation and their homogeneity. Other material could often, no doubt, be employed with advantage.
1. Preparation and Reaction of the Material
In a typical preparation, chicken erythrocytes were obtained from whole blood (about 200 ml) by centrifugation and washing in isotonic (0-93%) saline. Freezing and thawing has been found to be the most satisfactory method of preparation of uncontaminated nuclei. Five con
secutive thawings are required, and the temperature of the suspension must be kept below 5°. After four cold saline washes, to remove some attached stroma, the nuclei appeared in good condition, could be gelled, and smears fixed in alcohol showed normal Feulgen and BDC reactions and the water effect. The nuclei were suspended in cold saline (40 ml) and injected in a fine, rapid stream into cold, stirred ethanol (300 ml) avoiding formation of gross clumps. The fixed nuclei were washed with ethanol, and shaken in ethanol (360 ml) for 2 hr at room temperature. After two washes in acetonitrile, they were shaken for 4 hr in a flask containing dry acetonitrile (250 ml), benzoyl chloride (20-6 ml) and pyridine (14-1 ml).
Washes were given in alcohol (2), in 90%, 70%, 50% and 25%
alcohols and in cold water (3). The nuclei were stirred for 20 min in a cold solution of tetrazotized dianisidine (0-06% of the stabilized salt) in 2%
veronal buffer, p H 9 (360 ml) at 4°, and centrifuged. Washes (with centrifugation for 2 min only, at 1900 g) were given in veronal buffer, water, 0 · 05 N HC1 and water (2), followed by stirring for 15 min in 0 · 15%
R acid in 0 · 5% N a H C 03. All operations here were at 4°. The nuclei were then allowed to warm to room temperature for 15 min, while stirred in R acid solution. The product was washed with water (3), 1% N a2C 03 (3), 0 · 01 Ν NaOH and water (2). A marked difference in intensity of benzoyl-ated and parallel unbenzoylbenzoyl-ated, coupled nuclei is apparent after coupling.
The effects of variation in the washes at each stage have been examined to establish that the procedures are adequate. Thus, maximum attainable removal into the supernatant of triazenes and of excess
ACYLATION AND DIAZONIUM COUPLING 233 reagents has been demonstrated, in addition to the removal, by the prescribed washes, of adsorbed similar dyes added in test experiments.
2. Fractionation of Reacted Nuclei
The benzoylated, coupled nuclei were washed in ethanol-ether (3:1, 360 ml), removing about 4% of the total dye present. This material has the properties of a lipo-protein or a very highly benzoylated protein fraction. Repeated washes gave no further removal. Two similar extrac
tions of 10 min each under reflux were given, without further colour loss, to remove lipid. A preparation dried in vacuo at this stage yields about
1 · 9 g from 180 ml original blood.
Nucleic acids were extracted by N HC1 (3 batches) under reflux, the sulphonated dye-linked protein remaining insoluble. The release of material with an absorption peak near 260 πΐμ was followed (Table III), to show that nucleic acid removal was virtually complete in 20 min.
Trichloroacetic acid (5%) gives the same result but more slowly. No dye was lost in these removals.
234 Ε . Α . B A R N A R D
Extraction with 0·7 N NaOH (80 ml) under reflux, 12 min, gave an intensely red-orange solution, and an insoluble residue containing a very small percentage of the total dye. On titration, the solution changes to purple-blue at p H 10, and the dye-linked fraction is selectively precipi
tated around p H 6 to 5. Repeated re-precipitation freed it from some uncoloured proteins. The fraction obtained, containing, in covalent linkage, almost all the initial colour, is protein in nature, yielding on reduction, hydrolysis and chromatography a typical amino acid mixture, without purines, pyrimidines, sugars or any other obvious additional component.
A number of proteases, used singly or in combination, were ineffective in hydrolysing this material, presumably due to the benzoyl groups (Bergmann and Fruton, 1941) and dye groups present. Acid hydrolysis (6 N HC1) gave quite rapid destruction of the dye group, while reflux in 5 Ν NaOH gave slower, but still prohibitive, dye loss. Similar synthetic dyes alone show similar, though rather slower, destructions.
The solution finally found to the problem of cleavage of the peptide bonds with conservation of the azo links involved hydrolysis catalysed by a cationic detergent. Protein hydrolysis catalysed in dilute acid by anionic detergents has been shown by Steinhardt and Fugitt (1942) : the use of a cationic detergent in alkali was investigated here because of the affinity of such a detergent for the sulphonic groups in the dye radical, the lower sensitivity of these dyes to alkali, and the desirability of con
serving tryptophane. The best medium tried was 0 · 015 M cetyl trimethyl ammonium bromide (CTAB) in 0-3 Ν NaOH, under reflux, which gave very small dye loss and eventual complete hydrolysis of the protein.
4. Fractionation and Identification
The group(s) X linked to the dye radical in the tissue must be released on hydrolysis to give the dye compound IV.
3. Hydrolysis of the Azo-dye-linked Protein
C H30 ^ X — N = N — <f~\
OCH3 OHs SOoH reduction ( I V )
S03H
X . N H , +
H 03S SOoH
A C Y L A T I O N A N D D I A Z O N I U M C O U P L I N G 235 From the hydrolysate, the detergent-dye complex was salted out in strong alkali and dissolved in water. Ion-exchange chromatography (Zeo-Karb, 225/H+) with displacement by 0· 2 Ν NH4OH/50% ethanol, separated the dye compound. By paper chromatography of this and of the amino acids in the hydrolysate, the progress of hydrolysis was fol
lowed: 15-20 hr was sufficient for full release of IV. One main dye spot was obtained in several solvents, but no identification is possible by paper chromatography since a number of different bis-azo, sulphonated dyes of this type give very similar RF values.
On reduction by hydrosulphite, and chromatography, o-dianisidine and 1-amino-R-acid were identified (cf. equation 2) and an unknown compound (NH2-X, Table IV), positive to ninhydrin. Eluates of this unknown showed rising u.v. absorption below 240 m/x only, inconsistent with a tyrosine or tryptophane derivative.
Synthesis of 3-amino-tyrosine and 2-(or 4-)amino-histidine and comparison with N H2- X (Table IV) supported a derivation from histi
dine. The two isomeric amino-histidines are unstable (cf. Fargher and
T A B L E I V
P A P E R C H R O M A T O G R A P H I C B E H A V I O U R O F D Y E R E D U C T I O N P R O D U C T S
N i n D i a z o F l u o r e s ^max
C o m p o u n d (1) (2) RF (3) h y d r i n c o u p l i n g c e n c e (m/x)
N H2- X 0 · 18 0 · 10 0 · 3 4 + + _ < 2 0 0
A m i n o - h i s t i d i n e 0 · 18 0 · 10 0 · 3 4 + + — < 2 0 0
A m i n o - t y r o s i n e 0 · 16 0 · 3 0 + + B r i g h t 2 7 0
S o l v e n t s : ( 1 ) B u t a n o l : a c e t i c a c i d : w a t e r ( 4 : 1 : 5 ) (2) E t h a n o l : a m m o n i a : w a t e r ( 2 0 : 1 : 4 ) (3) P h e n o l : w a t e r ( 4 : 1 )
Pyman, 1919; Diemair and Fox, 1938; Hunter and Nelson, 1941) and were prepared together in solution only, by reduction of pure mono-(p-azo-benzoic acid)-histidine. I t appeared that one isomer only was obtained from the tissue derivative, but no orientation was possible.
Final confirmation was made by infra-red spectroscopic analysis, in collaboration with Professor W. C. Price, Mr. R. Bradbury, Dr. M. A.
Ford and Dr. G. R. Wilkinson. The compound NH2-X, obtained by elution after chromatography on thoroughly pre-washed paper, and deposited on an AgCl disc, gave an unusual infra-red spectrum (Barnard, 1957) showing interaction with groups in the cellulose, but similar spectra
236 Ε . A . B A R N A R D
were given by synthetic amino-histidine and by histidine itself (but not by tyrosine), after similar elution. This behaviour is given only after drying of the paper ; it appears that histidine and its derivatives then form a spectroscopically distinct complex with a cellulose fraction, presumably related to the strong hydrogen-bonding properties of the system (Barnard and Stein, 1958). Elution in the original wet state prevented this pheno
menon. However, the material then darkened during elution and depo
sition, and gave a different spectrum, containing bands characteristic of an amino acid, but suggesting self-polymerization had occurred (Barnard, 1957). Decomposition is minimized by chromatography in the dark in a reducing atmosphere, but still occurs in the solid state and when irradi
ated. The synthetic amino-histidine gave an identical spectrum,, not obtained from any other source.
Thus, although two types of anomalous behaviour were found in the infra-red study due to peculiarities of histidine, both were shown in the synthetic and tissue-derivatives, and these produced identical spectra in each case. The analyses left no doubt that X is histidine.
V. CRITIQUE OF THE METHOD