[4] Synthesis and use of colloidal gold-coupled receptor ligands

36

PREPARATION OF CHEMICAL PROBES

[4]

[4] S y n t h e s i s a n d U s e o f C o l l o i d a l G o l d - C o u p l e d Receptor Ligands

By LOTHAR JENNES, P. MICHAEL CONN, and WALTER E. STUMPF Great progress has been made in the past few years in identification and localization of chemical messengers, such as neuropeptides, in histological preparations. Immunohistochemical studies using specific antibodies against these peptides have helped to define their distribution in the central and peripheral nervous system. This approach often was limited to demonstration of sites of production, transport, and release but failed to identify the loci of action of the neuropeptides. This failure was usually caused by the small size of the neuropeptides, since most of their amino acids become unaccessable for the antibodies after the hormone was bound to a membrane receptor. Identification of sites of action of peptides thus relied on labeling the peptide directly and on subsequent incubation of tissue slices or cell cultures with the labeled hormone. Most commonly, radioactive isotopes, such as tritium or ~25Iwere used as label of peptides which were then detected autoradiographically. L2 Other approaches include the use of rhodamine labeling of the peptides which can be visualized in a fluorescence microscope with the aid of a videointensifying device 3 or conjugation ofa peptide to biotin which can be visualized by an incubation with avidin and horsereadish peroxidase with subsequent staining with 3,3'-diaminobenzidine-4 HCI. 4 In addition, peptides can be conjugated to ferritin5 or to horseradish peroxidase. 6 Depending on the goal of the study, a certain approach of visualization of the peptide has to be selected and each of the above ways has a drawback and limitations with regard to exposure times, precision of localization, fading of the marker, and resolution or preservation of the histological integrity of the tissue. Recently, colloidal gold has become generally used as a label for proM. J. Kuhar, in " M e t h o d s in Chemical N e u r o a n a t o m y " (A. Bjorklund and T. Hokfelt, eds.), p. 398. Elsevier, Amsterdam, 1983. 2 M. Duello, T. M. Nett, and M, G. Farquhar, Endocrinology 112, 1 (1983). 3 E. Hazum, P. Cuatrecasas, J. Marian, and P. M. Corm, Proc. Natl. Acad. Sci. U.S.A. 77, 6692 (1980). 4 L. Jennes, 1). Bronson, W. E. Slumpf, and P. M. Conn, Cell 17ssue Res. 239, 311 (1985). 5 C. R. Hopkins and H. Gregory, J. Cell Biol. 75, 528 (1977). 6 R. B. Dickson, J. C. Nicolas, M. C. Willingham, and 1. Pastan, Exp. Cell Res. 132, 488 (1981).

METHODS IN ENZYMOLOGY. VOL. 124

Copyright ~v 1986by Academic Press. Inc. All rightsof reproduction in any form reserved.

[4]

COLLOIDAL GOLD-COUPLED RECEPTOR LIGANDS

37

teins, including antibodies, staphylococcal protein A , 7-13 o r peptide hormones, such as insulin and gonadotropin releasing hormone. ~4-~vColloidal gold is especially well suited for electron microscopic studies, since it is electron dense and can be easily sized over a wide range, thus allowing simultaneous detection of multiple compounds.IS-2° Chemistry of Colloidal Gold Colloidal gold is formed by a reduction of gold chloride into complexes that are negatively charged on their surface. The negative charge seems to be caused by a dissociation of H[AuC12] into H + and AuCI2- at the surface of the particle or by the charges of an ionic cloud adsorped to the surface of the colloid. 2j Highly dispersed material is, under most conditions, unstable in aqueous solutions since the particles tend to aggregate or to adsorb themselves in order to reduce, under loss of energy, their surface areas and with it the number of free valences, thus achieving an energetically more favorable status. In the case of colloidal gold, however, all electrical surface charges are negative, which results in the generation of a strong electrical field. Since each gold particle creates its own electrical field, repulsion forces are generated which prevent an aggregation of the individual particles and are thus responsible for the stabilization of the sol. Since the relative surface area and with it the electrical charges of gold particles in a sol increase with decreasing diameter of the individual grain, it follows

7 W. P. Faulk and G. M. Taylor, lmmunochemistry 8, 1081 (1971). s E. L. Romano, C. Stolinski, and N. C. Hughes-Jones, Immunoehernisto' 11,521 (1974). 9 E. L. Romano and M. Romano, lmmunochemistry 14, 711 (1977). 10 W. D. Geoghegan and G. A. Ackerman, J. Histoehem. Cytochem. 25, 1187 (1977). i~ M. Horisberger and J. Rosset, J. Histochem. Cytochem. 25, 295 (1977). 12 N. D. Tolson, B. Boothroyd, and C. R. Hopkins, J. Microsc. 123, 215 (1981). 13 j. Roth, M. Bendayan, and L. Orci, J. Histochem. Cytochem. 28, 55 (1980). t4 G. A. Ackerman and K. W. Wolken, J. Histochem. Cytochem. 29, 1137 (1981). 15 R. B. Dickson, M. C. Willingham, and I. Pastan, J. Cell Biol. 89, 29 (1981). 16 D. A. Handley, C. M. Arbeeny, L. D. Witte, and S. Chien, Proc. Natl. Acad. Sci. U.S.A. 78, 368 (1981). t7 L. Jennes, W. E. Stumpf, and P. M. Conn, Endocrinology 113, 1683 (1983). ~s M. Horisberger, in "Scanning Electron Microscopy" (O. Johari, ed.}, Vol. 2, p. 9. SEM Inc., A. M. F. O'Hare, Chicago, 1981. ~" J. M. Lucocq and J. Roth, in "'Techniques in lmmunocytochemistry" (G. R. Bullock and P. Petrusz, eds.}, Vol. ill, p. 155. Academic Press, Orlando, 1985. 2o M. Horisberger, in "Techniques in Immunocytochemistry" (G. R. Bullock and P. Petrusz, eds.), Vol. III. Academic Press, Orlando, in press, 1985. zt W. Pauli, "Colloid Chemistry of the Proteins." Blakiston's, Philadelphia, 1922.

38

PREPARATION OF CHEMICAL PROBES

[4]

that colloidal gold preparations with small particle sizes are more stable than preparations with large particle s i z e s . 22 Since electrical repulsion due to the negative surface charge is the major force responsible for stability of the sol, colloidal gold is very sensitive to the addition of ions. Flocculation of colloidal gold is a process which is reversible only in its initial phase. According to Pauli, '-3 flocculation is caused by inactivation and association of opposingly charged ions, by interactions of the different neutral particles among each other which results in formation and decomposition of larger aggregates, and by a disproportioning of the separated auro-chloro complexes to Au, HCI, and H[AuCI4]. Ions with a charge opposite to the surface charge of the colloidal gold particle will eliminate the repulsion forces and induce aggregation and flocculation of gold particles. The amounts of electrolytes necessary to induce flocculation depend upon the valences of the particular ions. In order to eliminate the same amount of negative surface charge of colloidal gold and to induce flocculation, cations, such as K +, Ba >, and AP* have to be added in equivalent amounts of 1000:10:l to cause the same effect. 24 Protection of the hydrophobic colloidal gold against flocculation can be achieved by attaching macromolecules or peptides to the negatively charged surface of the gold particles, which causes a transformation of the characteristics of conjugate into those of a hydrophilic colloid. The process of adsorption, which is a noncovalent binding caused by Coulomb forces and by Van der Waal forces, is dependent on different factors such as pH, ionic strength, concentration, temperature, or electrolytes. Most proteins and peptides studied to date form a monolayer on the surface of the adsorbent and retain their original conformational characteristics as in aqueous solution. Since they are also attached at only one (for inflexible molecules) or a few sites (for flexible molecules), the majority of colloidal gold-peptide or gold-protein conjugates retain their biological activity. 2%2~' Depending on the size and flexibility of the attached peptide or protein, the protection of the colloidal gold is not complete and flocculation of the colloid may still occur after addition of salts. A more complete protection can be achieved by adding flexible polymers, such as, polyethylene glycol (PEG) with a molecular weight of about 20,000. According to = G. Frens, Kolloid-Z. Z. Polymere 250, 736 (1972). 23 W. Pauli, Heir. Chim. Acta 32, 795 (1949). 24 A. F. Holleman and E. Wiberg, " L e h r b u c h der A n o r g a n i s c h e n C h e m i e . " De Gruyter, Berlin, 1971. 2~ W. Heller and T. L. Pugh, J. Polymer Sci. 47, 203 (1960). 26 A. Silberberg, J. Phys. Chem. 66, 1884 (1962).

[4]

COLLOIDAL GOLD-COUPLED RECEPTOR L1GANDS

39

Heller and Pugh, 25 PEG is adsorbed to colloidal gold only at few sites while the remaining portions of the molecule can extend into the surrounding medium. These unbound portions of the polymers are responsible for "steric protection" of the colloidal gold particles, due to their identical charge. Based on mutual repulsion, the unbound polymers generate sufficient forces so that interpenetration of two PEG chains occurs only very limited. This way, the coated colloidal gold particles are kept at distances which are too large for the attractant Van der Waal forces to become effective. Sizing of Colloidal Gold Particles Preparation of colloidal gold particles is based upon reduction of HAuCI4 and is possible via a wide variety of reagents, including formaldehyde, white phosphorus, citric acid, ascorbic acid, tannic acid, or hydrogen peroxide (for review see Zsigmondy27; Ostwald28; see also Roth29). The principal chemical reaction has been known for over a century (Faraday 3°) although the colloidal nature of gold has not been recognized until 40 years later (Zsigmondy3~,32). The size of the individual gold particles can be easily manipulated and depends on several factors, such as the chemical nature of the reducing reagent, temperature, pH, or concentration of the reagents. In the following, five standard methods are described which allow the preparation of colloidal gold particles with a final diameter ranging from 3 to 150 nm. Some methods of synthesis, particularly of small diameter colloidal gold particles, use powerful oxidizing and reducing agents and the user should be familiar with the appropriate precautions and good laboratory practice. Preparation o f Colloidal Gold with an Average Si,~e o f 2-3 nm

This method was first described by Zsigmondy27.32 and utilizes white phosphorus as a reducing reagent. More recent descriptions of this technique can be found in Mace et al., 33 Roth, 34 or Jennes et al. 17 27 R. Z s i g m o n d y , " T h e C h e m i s t r y of Colloids." Wiley, New York, 1917. 25 W. Ostwald, "Practical Colloid C h e m i s t r y . " Methuen, London, 1926. 2~ j. Roth, in " T e c h n i q u e s in l m m u n o c y t o c h e m i s t r y " (G. R. Bullock and P. Petrusz, eds.), Vol. II, p. 217, A c a d e m i c Press, London, 1983. ~ M. Faraday, Phil. Trans. R. Soc. London 147, 145 (1857). q R. Zsigmondy, Liebigs Ann. 301, 31) (1898). ~2 R. Zsigmondy, Zttr Erkenntnis der Kolloide 100 (1905t. ~ M. L. Mace, Jr., N. T. Van, and P. M. Corm, ('ell Biol. Int. Reports I, 527 (1977). s4 j. Roth, in " T e c h n i q u e s in I m m u n o c y t o c h e m i s t r y " (G. R. Bullock and P. Petrusz, eds.), Vol. I, p. 107. A c a d e m i c Press, L o n d o n , 1982.

40

PREPARATION OF CHEMICAL PROBES

[4]

Ten milliliters of 0.1% (w/v) stock solution of aqueous chloroauric acid [H(AuCI4) • 4H20], which was stored protected from light, is added to 90 ml double distilled water and the pH is brought to 7.2 with 0.2 M potassium carbonate. The solution is heated under constant stirring and, at the boiling point, 0.5 ml diethyl ether with white phosphorus (1 part white phosphorus-saturated diethyl ether and 4 parts pure diethyl ether) is added. The color of the solution changes immediately to dark red and upon continued boiling to orange red. After 7 to 10 rain boiling, the reduction is complete and the sol can be cooled to 4°. If the sol is not stabilized immediately via PEG or attachment of proteins, the pH is adjusted to 6.9 with 0.1 M hydrochloric acid or acetic acid in order to allow longer storage of the colloidal gold. The pH of unstabilized or not fully stabilized colloidal gold should be measured either with a gel-filled combination electrode or with indicator paper since the electrodes of regular pH meters will be rapidly clogged by the charged gold particles. If a regular pH meter has to be used the sol should be stabilized with PEG, 5 to 10 rain before determination of the pH.

Preparation o f Colloidal Gold with an Average Size of 5 - 7 nm This method uses simultaneously the two reducing reagents tannic acid and citric acid, both of which were applied separately in earlier studies to produce colloidal gold of larger sizes. 28,35 According to Muhlpfordt, 36 10 ml of 0.1% stock solution of aqueous chloroauric acid and 90 ml of double distilled water are mixed in a 500-ml Erlenmeyer flask and heated under constant stirring to the boiling point. After 6.5 min boiling, 2 ml of 1% (w/v) sodium citrate, dihydrate solution and 0.45 ml of a freshly prepared solution of tannic acid (1% w/v) are rapidly and simultaneously added to the boiling solution. The color of the solution changes first to violet and under continuing boiling to wine red. After 5 rain of boiling the reduction is complete and the colloid can be cooled to 4° for further manipulation (Fig. I). The average gold particle size (95%) is 5.7 nm and ranges between 3 and 8.4 nm. The size of the particles can be increased to 8.5-15 nm by changing the conditions of the reduction, i.e., if the tannic acid concentration is increased or decreased, the tannic acid-citric acid mixture is added slowly or has been boiled before use, or if the volume of the flask is changed. 36 35 G. Frens, Nature (London) Phys. Sci. 241, 20 0973). 36 H. Muhlpfordt, Experientia 38, 1127 (1982).

[4]

COLLOIDAL

GOLD-COUPLED

RECEPTOR

41

LIGANDS

1 t

"

°

*"

,.

"t . o

p.

" :

•

•

..

.

%.

,. ..

t

.

. * .

~

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,

,

-

-

% 2

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4P

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41 5

Fn(~s. 1-5. Different sizes of colloidal gold ranging from 5 nm (Fig. 11 to 16(1nm (Fig. 5). Nonpurified colloidal gold prepared after the method of Muhlpfordt ~' (Fig. I) or of Frens ~ (Figs. 2-5). Amount of sodium citrate was reduced from 2 ml of a I% solution (Fig. 2) to 1.0 ml (Fig. 3), to 0.7 ml (Fig. 4), and to 0.4 ml (Fig. 5). Magnification: ×42,000 (bar 200 nm).

Preparation of Colloidal Gold with an Average Size of 5-12 nm T h e d i s p e r s i o n p r o c e d u r e is s i m i l a r to t h e o n e d e s c r i b e d for the p r e p a r a t i o n o f c o l l o i d a l g o l d w i t h t h e size o f 2 - 3 nm a n d is a l s o b a s e d u p o n Zsigmondy's original description? 2 T w o a n d o n e - h a l f milliliters o f 0.6% (w/v) a q u e o u s s o l u t i o n o f c h l o r o a u r i c a c i d is m i x e d with 60 ml d o u b l e distilled w a t e r a n d 0.5 ml o f d i e t h y l e t h e r p h o s p h o r u s (as a b o v e ) is a d d e d at r o o m t e m p e r a t u r e . U n d e r c o n t i n u o u s s t i r r i n g f o r 15 rain at r o o m t e m p e r a t u r e the m i x t u r e c h a n g e s its c o l o r to b r o w n . T h e s o l u t i o n is t h e n h e a t e d a n d k e p t a b o v e the boiling p o i n t f o r 5 rain. D u r i n g this t i m e t h e c o l o r c h a n g e s to w i n e red. A f t e r

42

PREPARATION OF CHEMICAL PROBES

[4]

completion of the reduction the colloidal gold is cooled to 4 ° for further manipulation.

Preparation of Colloidal Gold with an Average Size of 8-15 nm Controlled reduction of chloroauric acid with ascorbic acid leads to colloidal gold particles with a range in size between 8 and 15 nm. 37 Ten milliliters of a 0. 1% stock solution of HAuC14 and 1 ml of a 0.1 M K2CO3 solution are added to 15 ml distilled water. The mixture is cooled on ice before I ml of a 0.7% solution of sodium ascorbate is added under constant stirring. The color changes immediately to dark red and after addition of 75 ml distilled water, the mixture is heated to the boiling point in order to complete the reaction. After 5 min boiling, the color becomes lighter and the sol can be cooled to 4 ° . Higher temperatures during the reduction of the chloroauric acid were reported by some authors to lead to an increase in the size of the gold particles, 38 although Horisberger and Tacchini-Vonhagen 39 prepared colloidal gold of 11.6 -+ 2.4 nm at room temperature which is almost identical to the size prepared by Slot and Geuze 3~ at 4 ° (11.3 + 35%).

Preparation of Colloidal Gold with an Average Size between 16 and 150 nm The most versatile procedure to prepare colloidal gold particles is with trisodium citrate as reducing reagent. Depending upon the amount of citrate added to an identical amount of chloroauric acid, the size of the gold particles can be manipulated in a controlled and reproducable way between 16 and 150 rim. 35 Ten milliliters of a 0. I% stock solution of HAuC14 is added to 90 ml of distilled water and heated to the boiling point. Under constant stirring 2 ml of a 1% solution of citric acid, trisodium salt dihydrate is added to the gold solution and the mixture is kept for another 5 rain above the boiling point. The solution changes its color initially to blue and after about 2 rain continuous boiling to red. After an additional 3 min boiling, the sol is cooled to 4 ° for further processing. If a larger size of the colloidal gold particles is anticipated, the amount of citrate added to an identical HAuC14solution is reduced. According to Frens, 35 addition of I ml of a 1% solution of Na3 citrate to 100 ml HAuC14 results in gold particles with an average size of 41 nm, 0.42 ml of 1% 37 F. C. Stathis and A. Fabikanos, Chem. Ind. (London) 27, 860 (1958). 38 j. W. Slot and H. J. Geuze, J. Cell Biol. 90, 533 (1981). 39 M. Horisberger and M. Tacchini-Vonlanthen, Histochemisto, 77, 37 (1983).

[4]

COLLOIDAL GOLD-COUPLED RECEPTOR LIGANDS

43

solution of citrate will produce gold particles of about 97 nm, and 0.32 ml citrate will generate gold particles of about 150 nm average size. If larger (40-150 nm) sizes of the gold particles are to be prepared, the boiling time should be extended to up to 30 rain in order for the reductions to be complete (Figs. 2-5). Conjugation of Peptides to Colloidal Gold As discussed earlier in this chapter, peptides or proteins can be linked to the negatively charged colloidal gold particle. The amount of peptide attached to the individual particle depends upon a variety of factors including the size of the gold particle, the pH at which the coupling is performed, the concentration of the peptide added, and the electrical charge of the peptide. 4°,41 Coupling takes place only if the negative charge of the unstabilized colloidal gold can be compensated by a net positive charge of the peptide. Due to the lability of unstabilized gold and its sensitivity to salts, only salt-free peptide or protein preparations should be used for the coupling. The point of maximal stabilization of the gold particles by a protein or peptide, i.e., the point at which the sol is protected from salt-induced precipitation, is usually determined according to the procedure of Horisberger and Rosset. j~ Small amounts of unstabilized colloidal gold (0.5-1 ml) are added to a series of increasing concentrations of related protein in a constant volume (100 p~l). After 5 min, 100 /xl of a 10% NaC1 solution is added to the mixture. If the color changes from red to violet and finally to blue, the protection was incomplete and aggregation and subsequent flocculation of unstabilized gold was induced by the added salt. Spectrophotometric analysis of the colloidal gold at 520 nm results in a more precise estimate of the degree of stabilization of the sol. For most peptides or proteins, a final concentration of 10% above the "optimal" amount is chosen to stabilize the gold. After the optimal conditions for the adsorption have been characterized, the unstabilized gold sol (100 ml) is added under constant stirring at room temperature to the peptide. After 5 rain, 5 ml of I% PEG is added, and the pH is adjusted. The conjugate can now be stored for longer periods of time at 4 °. In order to obtain evenly coated gold particles especially of larger sizes, it appears to be important that the gold sol is added to the peptide and not vice versa. 1~,41 4oS. L. Goodman, G. M. Hodges, L. K. Trejdosiewicz,and D. C. Livingston,J. Microsc. 123, 201 (1981). 41M. Horisbergerand M. Vauthey,Histochernisto, 80, 13 (1984).

44

PREPARATION OF CHEMICAL PROBES

[4]

The main goal of the above method is to stabilize the gold via attachment of large amounts of proteins. This method is commonly used in immunohistochemical procedures where gold is coupled to protein A or immunoglobulin. Depending upon the compounds and experiments studied, a maximal load of gold particles with protein is not always desired, since this reduces sensitivity. The amounts of protein attached to the gold under "optimal and suboptimal" conditions can easily be monitored if radioactive tracer is added to the peptide to be labeled. In our experiments with labeling GnRH-agonists (D-Lys~'-GnRH) or antagonists (D-pGIu J-D-Phe2-D-TrpJ-D-Lys6-GnRH and N-Ac-D-p-CI-Phel.2,D-Trp3-D Lys6-D-AIaI°-GnRH) we did not observe indications which would require maximal load of the colloidal gold particles with these analogs nor were we able to fully stabilize the gold by the addition of peptide only. Probably due to the small size of the peptide which may not produce sufficient "steric protection," addition of PEG was always required in order to stabilize the gold. According to our protocol 10 ml of unstabilized colloidal gold with 20 /xm particle size is added to 0.1, 1, 10, or 50/zg analog and the attachment is allowed to continue for 10 min at room temperature. After this time, the sol is stabilized with PEG (100 mg/ml) and can be kept after sterilization by Millipore filtration (0.2/zm) for at least 1 month at 4° without noticable decrease in activity. Before use, we centrifuge the conjugate at 4° for 60 min at 100,000 g (particle size of 3-12 nm), 40,000 g (particle size of 10-20 nm), or 20,000 g (particle size larger than 20 nm). The pellet is then resuspended in the appropriate medium. Several factors which influence the attachment and the biological activity of the conjugate need to be characterized. The p H at which the attachment is conducted has been suggested to have a great influence on the amount of protein bound and therefore on the stability of the gold conjugate if no PEG is added. 10Most reports show that a pH near or slightly above the isoelectric point of the protein gives the most favorable results. Most of these experiments were carried out with large proteins, such as albumins, horseradish peroxidase, or protein A. We can, however, not demonstrate this pH dependence for the conjugation of the above GnRH analogs. Judging from the amount of J25Ilabeled GnRH analog attached to colloidal gold of 8 or 20 nm we were unable to detect significant differences in the amount of bound hormone if the coupling was performed at a pH between 4 and 8. There was a slight trend which indicated an increase of adsorption at a pH above 8.5. In our experiments, we used, however, submaximal amounts of GnRH and achieved a 50-80% efficiency of the attachment. The biological activity of the conjugate needs to be established. This is especially important for studies on receptor-mediated endocytosis. When

[4]

COLLOIDAL GOLD-COUPLED RECEPTOR LIGANDS

45

smaller ligands (5 to 20 amino acids) are used in amounts which require addition of PEG, steric hindrance due to the larger PEG molecule may occur and reduce or even abolish the biological activity of the ligandcolloidal gold conjugate. 41 Besides steric hindrance, the site in the molecule of the ligand at which the gold is attached, can be important for continued biological activity. This problem may be difficult to overcome if the ligand is a large, flexible molecule which is attached to the gold at several sites depending upon the presence of electrically positive charged groups (for example, e-aminoimidazol). However, if small peptides are used as a ligand, the probable site of attachment can be influenced by using synthetic analogs which have suitable amino acid substitutions. In our experimental system analogs to GnRH are used which have a D-lysine substitution in position 6. Since coupling is executed at a high pH and the e-amino group of the lysine is the highest charged reative group in the molecule under these conditions, attachment is likely to occur at this site. The conformation of GnRH in solutions resembles a twisted U with the carboxy- and the N-terminus close together. This conformation is required for binding to the GnRH receptor and for inducing a biological response. Since most peptides form an even monolayer on the surface of the colloidal gold particles and do not accumulate in clusters 42 it can therefore be expected that attachment in the "remote" position " 6 " does not interfere with binding and the biological activity. Our data on the physiological activity of the GnRH-colloidal gold conjugates support this view. Thus, D-Lys6-GnRH-colloidal gold retains on a molar basis 90% of its agonistic activity when compared with free agonist, o-p-Glu~-o-Phe 2D-Trp3-D-Lys6-GnRH-colloidal gold retains 79% of its antagonistic activity and N-Ac-D-p-C1-Phel,2-D-Trp3-D-Lys6-D-Alal°-GnRH-colloidal gold is active as an antagonist with an efficacy of 80%. The stability of the colloidal gold-protein conjugates has repeatedly been a matter of concern with regard to a possible dissociation of the gold and the protein and with regard to a flocculation of the gold over time. More reports in the literature, however, indicate that a detachment of ligand and colloidal gold does not represent a serious problem if the proper precautions are met. Thus, after labeling colloidal gold (40 nm diameter) with "optimal ''~j amounts of ~25I-labeled concanavalin A and subsequent stabilization with PEG, followed by 3 wash/centrifuge cycles, only 1.7% of the total radioactivity was recovered in the supernatant 2 days after the initial coupling. No further detachment was observed during the following 45 days. 43 Using "suboptimal" amount of the ~2~I-labeled 42 F. R. Eirich, J. Colloid Interface Sci. 58, 423 (1977). 4~ M, Horisberger and M. Tacchini-Vonlanthen, in - L e c t i n s ' " IT. L. Bog-Hansen and G. A. Sprengler, eds.), Vol. 111, p. 189. l)e Gruyler, Berlin, 1983.

46

PREPARATION OF CHEMICAL PROBES

[4]

GnRH antagonist N-Ac-D-p-C1-PheL2,D-Trp3-D-Lys6-D-AlaJ°-GnRH, followed by PEG stabilization, we did not detect any detachment over a period of 20 days even when the conjugate was kept at room temperature. An influence of the initial concentration of adsorped protein on the degree of dissociation from the gold particles at early time points was observed for ~2~l-labeled bovine serum albumin. 44 An increase in the amount of initial protein (1 mg protein/ml sol) resulted in an increase of dissociated protein during the first 4 hr in a linear, concentration-dependent way. Detachment was, however, negligible if the amount of the initial albumin was low (0.01 mg/ml sol). This phenomenon did not change upon addition of PEG. 44 From our experience and from data in the literature it appears that properly stabilized colloidal gold-peptide complexes can be kept for a long period of time without significant reduction in this activity assuming that the peptide itself is stable. For the protection of the sol against microorganisms, the conjugate should be Millipore filtered and, if the experimental conditions allow, sodium azid can be added to a final concentration of 0.1%. In order to ensure that no large gold aggregates or free ligand are present in the experimental mixture we always centrifuge the conjugate first at low speed (1000-20,000 g) and the resulting supernatant at high speed (20,000-100,000 g ) j u s t prior to use. The pellet obtained in the second centrifuation is resuspended in the appropriate medium and used within the next few hours.

The Purification of Colloidal Gold-Protein Conjugates A narrow range of variations in the size of the colloidal gold-ligand conjugate is of great importance if several substances are to be visualized in the same histological preparation. The selection of the reducing reagent and the conditions of the chemical reaction will largely determine the sizes of the individual particles; however, even under optimal conditions, a variation in the size of the particles over 15-30% can be expected. In order to obtain more homogeneous populations of conjugate, additional purification is commonly performed, using differential centrifugation through a discontinuous sucrose gradient with concentrations between 5 and 30%. 38 Small gold particles (3-6 nm) are centrifuged for 45 rain at 280,000 gmax (41,000 rpm in a Beckman SW 4! rotor), medium size particles (8-12 p~m) for 30 min at 70,000 gma× (20,000 rpm), and larger size particle (< 15 nm) for 30 rain at >20,000 gnl,~x(> 10,000 rpm). If a sucrose 44 j. B. Warhol, R. Brelinska, and D. C. Herbert, Histochemistry 76, 567 (1982).

[5]

BIOTINYLATED NEUROENDOCRINE PEPTIDES

47

gradient is used, the colloidal gold should be fully stabilized before the centrifugation. As an alternative, an aqueous glycerol gradient over 1030% concentrate can be used which offers the opportunity to stabilize or conjugate the colloidal gold to protein after purification of a homogeneous population of gold particles. In addition, colloidal gold and its conjugates can be stored in high (50%) concentrations of glycerol at low (-18 °) temperatures.3S Acknowledgment Research supported by NIH Grants NS17614 and HD19899.

[5] P r e p a r a t i o n a n d U s e o f B i o t i n y l a t e d Neuroendocrine Peptides

By ELI HAZUM The high affinity constant (10 -j5 M) between the glycoprotein avidin and the vitamin biotin provides an important experimental tool for a wide variety of biological applications. The avidin-biotin complex has been used as a mediator in localization, isolation and immunological studies (reviewed in refs. 1-4). Recently, biotinylated peptide hormones have been used for the localization and isolation of receptors on cell surfaces. For this purpose, biotinylated forms of various hormones have been prepared, e.g., corticotropin-releasing factor: adrenocorticotropic hormone, 6,7 human chorionic gonadotropin, 8 glucagon, 9 enkephalin, ~° and E. A. Bayer and M. Wilchek, Methods Biochem. Anal. 26, I (1980). 2 E. A. Bayer, E. Skutelsky, and M. Wilchek, this series, Vol. 83, p. 195. 3 G. V. Childs, in "Immunocytochemistry" (P. Petrusz and G. Bullock, eds.), p. 85. Academic Press, New York, 1983. 4 M. Wilchek and E, A. Bayer, lmmunol. Today 5, 39 (1984). 5 K. N. Westlund, P. C. Wynn, S. Chmielowieo, T. J. Collins, and G. V. Childs, Peptides 5, 627 (1984). 6 K, Hofmann and Y. Kiso, Proc'. Natl. Acad. Sci. U.S.A. 73, 3516 (1976). v K, Hofmann, F. M. Finn, and Y. Kiso, J. Am. Chem. Soc. 100, 3585 (1978). 8 R. Riesel, E. A. Bayer, M. Wilchek, and A. Amsterdam, Isr. J. Med. Sci. 13, 968 (1977). 9 K. C. Flanders, D. H. Mar, R. J. Folz, R. D. England, S. A. Coolican, D. E. Harris, A. D. Floyd, and R. S. Gurd, Biochemistry 21, 4244 (1982). 10 A. Koman and L. Terenius, FEBS Lett. 118, 293 (1980).

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