Bismuth chemistry and the glutaraldehyde insensitive staining reaction

0040-8166/82/00030039

TISSUE & CELL 1982 14 (1) 39-46 ,Q 1982 Longman Group Ltd

$02.00

D. A. BRODIE

BISMUTH CHEMISTRY AND THE GLUTARALDEHYDE INSENSITIVE REACTION Key words:

Bismuth,

chemistry,

STAINING

staining.

ABSTRACT. The free metal ion, Bi a+, is the only chemical species of bismuth that stains a strongly bismuth-reactive molecule, polyarginine, in vitro. The bismuth solution specifically requires tartrate as a chelating agent for the reaction to occur reacts strongly with polyarginine, creatine and ATP between pH 7.4 and 8.0. Since Bi3+ fixed to cellulose acetate strips and DEAE-cellulose and P-cellulose, the free metal ion (Bia+) may bind to phosphate or guanidyl groups, or both, after glutaraldehyde fixation.

not a measure of its phosphate content (Brodie et al., 1982a, b) and bismuth does not stain nucleic acids (Locke and Huie, 1977a). The properties of the bismuth staining solution and its reaction to various test molecules in vitro have been investigated to suggest ways in which bismuth may bind to fixed cell components.

Introduction BISMUTH was originally used as a nonspecific section stain to show glycogen, lysosomes, ribosomes, and aldehyde groups (Ainsworth et al., 1972) and ferritin (Ainsworth and Karnovsky, 1972) and intensify uranyl and lead staining (Riva, 1974; Barrett et al., 1975). In contrast to section staining, en bloc staining by bismuth is highly specific (Locke and Huie, 1975, 1976, 1977a, b, 1980; Brown and Locke, 1978). The tissue staining by bismuth of some structures is sensitive to glutaraldehyde fixation. In vitro chemical tests with biological molecules spotted on to cellulose acetate strips showed that amino groups, but not phosphate groups, are sensitive to glutaraldehyde fixation (Locke and Huie, 1977a). However, electron spectroscopic imaging of the phosphorus content in a number of bismuth-reactive structures indicates that the intensity of bismuth staining of a structure is

Materials and Methods Preparation of poly-amino acid discs Zn vitro tests to assess the characteristics

of a stain are little used. Locke and Huie (1977a) developed a method for such testing which was modified for this work. Discs, 6 mm in diameter, were punched from cellulose acetate strips. Aqueous solutions of the polyamino acids and creatine obtained from Sigma Chemical Co. were loaded on to the discs with a 5 ~1 pipette and then fixed to the discs by overnight exposure to glutaraldehyde vapour. Preparation of the bismuth solution using diflerent chelators

The Cell Science Laboratories, Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7. Mailing address: The Cell Science Laboratories, University of Western Ontario, London, Ontario, Canada N6A 5B7. Received 28 September

The bismuth solution was made according to Locke and Huie (1977a) except that a 1: 2.5 dilution was made with 0.2 M triethanolamine-HCl (TEAM) buffer. The various

198 I. 39

40

BRODIE

chelating agents (sodium tartrate, sodium formate, oxalic acid, fumaric acid, malic acid, sodium citrate, and sodium ethylenediamine tetra-acetate) were made to equimolar concentrations.

8-

Bismuth reaction

Following fixation, the poly-amino discs were rinsed for 1 min in a 0.1 M TEAM buffer and reacted with bismuth for 1 min. The discs were then washed in 0.1 M TEAM as quickly as possible and immersed in an ammonium sulfide solution for 1 min. The discs were washed and cleared in Sepra Clear (Gelman Instrument Co.). They were then dried on to microscope slides on a slide warmer and photographed. The ion exchange material was washed in 0.1 M TEAM and reacted with bismuth for 1 hr. The material was then washed in 0.1 M TEAM and suspended in an ammonium sulfide solution. The material was subsequently washed in 0.1 M TEAM and photographed. Determination of metal reacfing species in solution

The metal solutions were made up with the following molarities: Bismuth Lead Nickel Aluminum

0.1 M O-1 M 0.7 M 0.7 M

in in in in

1.0 M HC104 1.0 M HC104 1.4 M HCl04 distilled water

These molarities were used to ensure that as the pH of the solution was raised, the solution would pass across the 1 ‘A borderline for polycomplex formation before precipitation could begin. The species of metal present in a solution depends upon the total metal concentration and pH (Kragsten, 1978). As the pH increases, the free metal tends to be hydrolysed and form complexes. This is usually shown graphically as the 1% borderline where only 1% of the metal is in polycomplex form (Fig. 1). As the pH is further increased, the metal hydroxides begin to precipitate when their limiting concentration is exceeded. This is usually shown as the point where precipitation begins (Fig. 1). Polyarginine discs (50 pg/disc) were processed as for the bismuth reaction described above using these metal solutions

2

4

6

Fig. 1. Hypothetical curves plex borderline (-.,.,) and the tation (-) for a metal concentration) as a function of

8

of the 1% polycombeginning of precipi(log of total metal pH.

at various pHs and the results recorded qualitatively. Preparation of ion exchange chromatography material

Cellulose-based ion exchange chromatography material was prepared according to Peterson (1970). Sephadex G-25 was suspended in distilled water overnight to permit swelling. All the chromatography material was washed several times in 0.1 M TEAM at pH 7.6 just before use. Results Binding of bismuth, lead, nickel, and aluminum to polyarginine in vitro

Bismuth will react strongly with polyarginine spotted on to cellulose acetate strips (Locke and Huie, 1977a; Fig. 2). Thus, polyarginine is a good test molecule for determination of the reactive form of bismuth. At a pH less than -1.0 where only free bismuth ions exist, a strong reaction is produced (Table 1). No reaction occurred at pH 3.4 or 12.0 where polynuclear complexes of bismuth or bismuth precipitates, respectively, predominate. Bismuth is a relatively heavy element (atomic number= 83) which readily forms polynuclear complexes and bears a charge of +3. To determine the considerations in the binding of bismuth to tissues, polyarginine discs were reacted with lead, nickel, and aluminum at various pHs. A strong reaction occurred at a pH between 5-O and 8.5 where the polynuclear

BISMUTH

CHEMISTRY

AND

41

REACTIVITY

b Fig. 2. Poly-amino acids and creatine were spotted on to cellulose acetate discs (50 pg/disc) and reacted with bismuth after overnight fixation in glutaraldehyde vapours as described in Materials and Methods. Only polylysine, polyarginine, and creatine reacted with the bismuth. a, solvent control; b, polyhistidine; c, polyasparagine; d, polylysine; e, polyarginine; f, creatine.

complex Pb4(0H)J4+ predominates (Maroni and Spiro, 1967) but there was no reaction where lead existed as Pb2t or where it precipitated (Table 2). Nickel and aluminum are not heavy elements (atomic numbers= 28 and 13, respectively) but they form polynuclear complexes, Ni4(0H)d4+ (Kragsten, 1978; Cotton and Wilkinson, 1980), and A11304(OH)24(Hz0)127+ (Johansson et al., 1960; Waters and Henty, 1977). Nickel reacts only weakly as a free metal ion or polynuclear complex and aluminum does not react at all with polyarginine. Thus the reaction with polyarginine is strong with metal species with a high atomic number, a valency of at least 3, and a relatively small size, particularly with regard to polynuclear complex formation. Chelation

specificity

in the bismuth reaction

Staining specificity can be improved by employing solutions of relatively low metal

concentrations (Hayat, 1975). Thus, it is beneficial to use a chelating agent which leaves just enough free metal in solution to react specifically with the tissue and not become a general tissue stain. Tartrate is used in the bismuth solution for just this purpose. According to Kragsten (1978), at a bismuth concentration of 25 mM and tartrate concentration of 55 mM, the 1 ‘A borderline for polycomplex formation is at pH 0.4 and precipitation begins at about pH 4.3. Yet bismuth is not precipitated at these concentrations at pH 7.6 which are used for staining. In an attempt to explain this discrepancy, on the basis of chelate type and/or ionization constants, bismuth was dissolved in equimolar amounts of several different chelating agents and the appearance of the solution monitored as the pH was raised (Table 2). Tartrate was the only solution which cleared at a pH above 4.3 when polycomplex formation should be prevalent. All other

Table 1. Metal binding to polyarginine in vitro as a function of complexation .-.-

~~

pH range at which:t

Metal Bismuth*

Molecular weight -.__209.0

Lead*

207.2

Nickel*

58.7

Aluminumrj

27.0

Free metal predominates Strong1 (pH< -0.5) None (pH< 3.5) Weak (PH<6.0) None (pH<2.5)

Polynuclear complex predominates

Metal precipitate predominates

None (l.O
None (pH> 5.0) None (pH>9.5) None (pH>7.5) None (pH>3.5)

* Precipitated with ammonium sulfide. t pH ranges taken from Kragsten (1978). $ Assessment made on the basis of colour (brown to black for sulfide precipitates and purple for hematoxylin precipitates). 5 Visualized as the hematoxylin lake.

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BRODIE

Table 2. Appearance of bismuth solution with various chelating agents as a result of changes in pH

_

Chelating agent

Ionization constants? ~.~_ Type*

P&

PKZ

PKZ

Solution appearance PH 0

pK4

Formate (Na)

Unidentate

3.77

Tartrate (Na)

Bidentate

4.16

2.89

Clear

Oxalate

Bidentate

4.14

1.25

Clear

Fumarate

Bidentate

4.54

3.03

Clear

Malate

Bidentate

5.20

3.40

Clear

Citrate (Na)

Tridentate

5.41

4.75

3.09

EDTA (Na)

Pentadentate or hexadentate

IO.26

6.16

2.67

Clear

* After Cotton and Wilkinson (1980). t After Jencks and Regenstein (1973). 1: Milky white indicates predominantly pr&ipitation of bismuth oxide.

Clear 2.00

polynuclear

solutions formed polycomplexes and/or precipitates. There was no correspondence with chelate type or ionization constants. There must therefore be a specific or selective reaction between bismuth and tartrate which prevents precipitation at pH 7.6 or higher. In vitro ligand specificity of bismuth following glutaraldehydefixation In vitro spot tests using biological molecules fixed to cellulose acetate indicate that the bismuth staining of many structures is sensitive to glutaraldehyde fixation (Locke and Huie, 1977a). A summary of these spot test results is shown in Table 3 and indicates that guanidyl, phosphate and/or the c-amino group of lysine may represent bismuth reactive groups insensitive to glutaraldehyde. To confirm and extend these spot test results, the bismuth reaction was carried out with reactive groups bound to ion exchange chromatography material. The results are shown in Figs. 3-10. Bismuth bound to DEAE- and P-cellulose very strongly and only weakly to CM-cellulose. It did not bind to Thiol-Sepharose or Sulfopropyl-Sephadex. Bismuth did remain in the Sephadex control material and since Sephadex bears no charge, the bismuth may have been caught in the pores and washed out only slowly. The

Clear

complex

pH 3-5 pH 7-8 Milky whitef Milky

Milky white Clear

Milky white Milky white Milky white Milky white Milky white

Milky white Milky white Milky white Milky white Milky white

formation

at pH 12 Yellow Clear Yellow Yellow Yellow Milky white Milky white

and yellow indicates

bismuth reactivity was not sensitive to glutaraldehyde fixation and it may be that the reactive groups of the chromatographic material are too far apart spatially to be effectively cross-linked. These results show that some amino groups (particularly guanidyi) as we!! as some phosphate groups are possible glutaraldehyde-insensitive bismuth reactive groups and sulfhydryl and sulfate groups are not. Discussion Bismuth chemistry In vitro spot tests with polyarginine indicate that the reactive form of bismuth is the free metal, Bi3+. The trivalent charge may be important since trivalent bismuth and the tetravalent polynuclear lead complex (Pb4(OH)d4+), but not the divalent lead ion, stain polyarginine. Since polynuclear complexes of bismuth, such as Bis(OH)l#+, Bi6O66+, Bis06(OH)s3+, Bis(OH)207+, Big(OH)z16+, and 1957, 1959; Tobias, Bis(OH)z$+ (Olin, 1960; Maroni and Spiro, 1966), generally have a higher valency than the bismuth ion but do not bind with polyarginine, the separation between charges on the metal species may also influence the stability of the bond between the metal ion and its ligand. A

BISMUTH

CHEMISTRY

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REACTIVITY

Table 3. Possible bismuth-reactive chemical groups as determined in vitro spot tests reported by Locke and Huie (1977a) and this paper Reactive groups DNA RNA Carboxyl Imidazole

Indole

SulfGuanidyl Amide hydryl Polyasparagine

PolyPolycysteine lysine

Amino

Phosphate

Molecule

DNA RNA

Polyglutamate

Reactivity following formaldehyde fixation ~._. Reactivity

None None

None

None

None

Strong

n.d.*

None

Strong Strong

n.d.

None

nd.

Strong

None

n.d.

Weak

nd.

nd.

Polyhistidine

Indole

Polyarginine

ATP

Weak

following glutaraldehyde fixation * n.d. = not determined. high atomic number seems necessary for detection because bismuth and lead stain polyarginine strongly and nickel and aluminum in any form do not. The bismuth solution is prepared by addition of sodium tartrate as a chelating agent to keep the free metal in low concentration and thus in solution. Tartrate forms soluble complexes with bismuth as either (BiTart& (Stary, 1963) or (BiTart)2+ (Chikryzova and Vataman, 1970). The results of this paper show that tartrate is the only chelating agent tested which can keep bismuth in solution at a pH between 7 and 8. There may be a specific or selective reaction

between bismuth and tartrate, just as for porphyrins and metals such as nickel, copper, lead, platinum, and iron (Martell and Calvin, 1952), that prevents bismuth precipitation. Glutaraldehyde-insensitive

bismuth Eigands

Test results for bismuth reactivity following glutaraldehyde fixation are summarized in Table 4. When biological molecules are spotted on to cellulose acetate strips, the bismuth reactivity of only those which bear a guanidyl, r-amino or phosphate group remains insensitive to glutaraldehyde. Ion exchange chromatography tests support this

Table 4. Summary of test results for bismuth reactivity following glutaraldehydefixation _~~ Test material Possible reactive group(s) Unlikely reactive group(s) Biological molecules spotted on to cellulose acetate strips*

Guanidyl, E-amino group of lysine, phosphate

DNA, RNA, carboxyl, imadozole, indole, amide, sulfhydryl

Ion exchange chromatography material

Amino, phosphate

Carboxyl, sulfate, sulfhydryl

Electron spectroscopic imaging of the beads in sitat

Phosphate

Not determined

* Locke and Huie (1977a) and this study. t Brodie et al. (1982a, b).

BRODIE

Figs. 3-10 Ion exchange chromatography material, which was unfixed (column A), fixed in 2.5 % glutaraldehyde (column B) or fixed in 2.5 % formaldehyde (column C), was reacted with bismuth and developed in ammonium sulfide. Fig. 3, cellulose; Fig. 4, CM-cellulose; Fig. 5, DEAE-cellulose; Fig. 6, P-cellulose; Fig. 7, Sepharose 4B; Fig. 8, Thiol-Sepharose; Fig. 9, Sephadex; Fig. 10, Sulfopropyl-Sephadex.

selectively. Electron spectroscopic imaging detects variable amounts of phosphorus in bismuth reactive structures which does not correlate with their bismuth affinity (Brodie et al., 1982a, b). Although some phosphate will not stain with the Bi3+ ion (for examde. nucleic acid phosphate, Locke and Huie, 1977a) it is also possible that bismuth binds

through guanidyl groups hyde fixation since bismuth polyarginine and creatine.

after glutaraldebinds strongly to

Acknowledgements I am grateful to Dr M. Locke for constructive criticisms and financial support. I would also

BISMUTH

CHEMISTRY

AND

REACTIVITY

like to thank Drs D. D. Perrin, R. J. Puddephatt and J. W. Lorimer for helpful discussions concerning bismuth chemistry.

45

This work was supported by Natural Sciences and Engineering Research Council grant A6607 to Dr M. Locke.

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STARY, J. 1963. Systematic TOBIAS, R. S. 1960. Studies

study of the solvent extraction of metal oxinates. Anal. Chim. Acta, 28, 132-149. on hydrolyzed bismuth (III) solutions. Part I E.m.f. titrations. J. Am. Chem.

Sm., 82, 1070-1072. WATERS, D. N. and HENTY, M. S. 1977. Raman

salts. J. C. S. Dalton, 1977, 243-245.

spectra

of aqueous

solutions

of hydrolysed

aluminum

(III)