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Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration
Immunochemistry, 1971,Vol.8, pp. 1175-1179. PergamonPress. Printedin Great Britain
C O M M U N I C A T I O N TO THE EDITORS Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration (Received It June 1971)
Enzyme labelled antibodies have been used cytochemically to detect antigens in tissues (Avrameas, 1970). With this technique, the most satisfactory results were obtained when peroxidase was employed as the enzyme label and glutaraldehyde was utilized to link the enzyme to the antibody (Avrameas, 1970, 1969). Although highly sensitive and capable of detecting small amounts of antigen, the immunoenzyme technique is limited by the penetration of the enzyme labelled antibody into the cell. This limitation is unimportant when organic fixatives like alcohol or acetone are used to fix the cells but it is particularly pronounced when cross-linking fixatives are used, like glutaraldehyde or formaldehyde, these being substances currently employed in electron microscopy. This paper presents a two step procedure which uses glutaraldehyde to prepare both peroxidase-antibody and peroxidase-Fab conjugates. The molar ratio of antibody or Fab to peroxidase is 1 : 1 and the conjugates can easily penetrate into the interior of well preserved formaldehyde fixed cells. The coupling of peroxidase to an antibody using a two step procedure via glutaraldehyde action is based on the observation that peroxidase alone could not be insolubilized even with an excess of glutaraldehyde (Avrameas and Ternynck, 1969). A possible explanation is that the free amino groups of the peroxidase molecule reacted with one of the two aldehyde groups of glutaraldehyde but that the configuration of the peroxidase derivative obtained was such that the second free aldehyde group was unable to combine with another peroxidase molecule. The free aldehyde group resting active should be available for combination with the amino groups of a protein added subsequently, providing that the two groups approach close enough. With this possibility in mind, we have tried adding antibody to the glutaraldehyde treated peroxidase under various experimental conditions. COUPLING OF GLUTARALDEHYDE TREATED PEROXIDASE WITH ANTIBODY OR Fab Sheep anti-rabbit IgG and anti-rat IgG antisera were prepared as described elsewhere (Avrameas et al., 1971). Sheep anti-rabbit Ig and anti-rat IgG antibody were isolated by passage of the whole antisera on an immunoadsorbent prepared by co-polymerization at pH 5 of 4 parts bovine serum albumin and one part IgG (Avrameas and Ternynck, 1969). The antibody preparations were subsequently filtered through Sephadex G-200 and the IgG containing fractions pooled for use. Fab-antibody fragments were prepared by the method of Porter (1959) using papain at a concentration of 2 per cent and a digestion o f 2 hr at 37°C. Fab and Fc fragments were separated from the non digested antibody by 1175
1176
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gel filtration of the reaction mixture (100 mg) through a Sephadex G-100 column (90 × 1.5 cm). The Fab fragment was then separated from the Fc component using a DEAE-cellulose column (20 × 2.5 cm) equilibrated with 0.0175 M phosphate buffer pH 6.9. Under these conditions, the Fab fragment is eluted in the breakthrough volume. Three different lots of horse radish peroxidase RZ3 were used, two from Boehringer, Mannheim (Germany) designated peroxidase PB1 and PB2 and one from Worthington, Freehold, New Jersey, designated peroxidase PW. To find the best conditions for the coupling of glutaraldehyde treated peroxidase with antibody, experiments were performed using buffers of several pH values and varying the concentrations of activated peroxidase and antibody. After a set of trials and taking into account the whole of the results obtained, the following technique was finally adopted. Ten mg of peroxidase were dissolved in 0.2 ml 0" 1 M phosphate buffer pH 6.8 containing 1.25 per cent glutaraldehyde (TAAB Lab., Reading, England). The solution was allowed to stand for 18 hr at room temperature and then filtered through a Sephadex G-25 column (60 × 0.9 cm) equilibrated with 0.15 M NaCI. The brown colored fractions containing the activated peroxidase were pooled and if needed concentrated to 1 ml with a Diaflo PM 10 membrane. To this solution, 1 ml of 0.15M NaC1 containing either 5 mg of antibody or 2.5 mg of Fab was added followed by the addition of 0.1 ml of 1 M carbonate-bicarbonate buffer pH 9.5. After 24 hr at 4°C, 0" 1 ml of 0"2 M solution of lysine was added and the mixture was then allowed to stand for an additional 2 hr. The preparation was then dialyzed at +4°C against several changes of buffered saline. The labelled antibody preparation was precipitated at +4°C with an equal volume of saturated neutral solution of ammonium sulfate. The precipitate was washed twice with half saturated ammonium sulfate solution, dissolved in a minimal volume of distilled water and exhaustively dialysed against buffered saline. The labelled Fab preparation was employed without treatment with ammonium sulfate. Both antibody or Fab peroxidase labelled preparations were centrifuged (20,1)00 g, 20 min) and stored at +4°C for 3 months without noting any loss in their capacity to detect the antigen. When a high proportion of peroxidase labelled antibody or Fab is wanted, as in the case of electron microscopic studies, 15 mg of peroxidase instead of 10 mg should be used. If wanted, peroxidase labelled antibody or Fab can be separated from free antibody or Fab by filtration of the preparation on a sephadex G-200 column (100 × 2 cm), although such treatment does not add any practical advantage for the detection of antigen. CHARACTERIZATION OF THE COMPOUNDS Chromatography of the three different lots of peroxidase on Sephadex G-200 column (100X2cm) calibrated for molecular weight determination gave a single peak corresponding to a molecular weight of 40,000. Chromatography of glutaraldehyde treated peroxidase PB1 and PB2 gave two peaks corresponding to molecular weights of 80,000 and 40,000, the former peak accounting for 23 per cent of the PB1 preparation and for 10 per cent of the PB2 preparation. The treated PW preparation gave a single chromatographic peak corresponding to a molecular weight of 40,000. Similar results were obtained when the three
Communications to the Editors
1 177
glutaraldehyde treated peroxidase preparations were run in sodium dodecyl sulfate (SDS 1%)-acrylamide (5%)/agarose (0"8%) gel electrophoresis (Shapiro et al., 1967; Uriel, 1966), and also when they were subjected to analytical ultracentrifugation. It thus appears that the treatment of peroxidase with glutaraldehyde produces a relatively low proportion of peroxidase dimers. When a mixture of antibody and peroxidase was applied to the above Sephadex G-200 column, two distinct chromatographic peaks were obtained; readings at 403 m/z revealing the chromatographic profile of peroxidase (Fig. 1A). When the peroxidase labelled antibody, prepared by the one step method previously described (Avrameas, 1969) and in the following termed method 1, was chromatographed only one asymmetric peak was obtained which emerged with the void volume of the column (Fig. 1B). With the peroxidase labelled antibody prepared by the present two step method, in the following termed method 2, also only one asymmetric peak was obtained (Fig. 1C). The readings at 403 m/z showed I 0.150
01
i
i i i i i i
0,100
0.050
i
0.150
® I~ 0.100
d 0.050
i
OJSO
© 0,100
0.050
20
40 60 TUBE NUMBERS
80
Fig. l. Chromatography on Sephadex G-200 column (100x 2 cm) equilibrated with 0-1 M phosphate buffer p H 6.8 o f (A): a mixture of sheep antibody anti-rabbit Ig and peroxidase: (B): peroxidase labelled sheep antibody anti-rabbit Ig prepared by the one step procedure; (C): peroxidase labelled sheep antibody anti-rabbit prepared by the two step procedure. ( ) optical density at 280 m/~ (. . . . . ) optical density at 403 m/z.
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Communications to the Editors
peroxidase eluted just before the position of IgG and corresponding to a molecular weight higher or equal to 200,000 while the reading at 280 m g showed a maximum in the position of IgG (Fig. 1C). The peroxidase labelled antibody preparations obtained by method 1 and method 2 were subjected to SDS-acrylamide/agarose gel electrophoresis. The method 1 labelled antibody hardly penetrated the gel. With the method 2 labelled antibody, two main bands were noted; one had a molecular weight of approximately 170,000 corresponding to free antibody and the other to a molecular weight of 210,000 corresponding to antibody-peroxidase conjugates; the latter was stained using the specific substrate for peroxidase. Similar experiments were carried out with peroxidase labelled Fab prepared by methods 1 and 2. Coupling by method 2 gave rise to two main fractions. The first has a molecular weight of 80,000 and the second of 40,000. In the 80,000 molecular weight fraction, Fab-peroxidase conjugates were found and, when peroxidase PB1 or PB2 were used, dimers of peroxidase molecules. In the 40,000 molecular weight fraction, free peroxidase was found and, when the quantity of peroxidase employed for the coupling was low, free Fab. Coupling of peroxidase to Fab by method 1 gave rise to a highly heterogeneous population of conjugates having a molecular weight range from 80,000 to 160,000. EFFECTIVENESS OF THE CONJUGATES TO DETECT INTRACELLULAR ANTIGEN Experiments were carried out to determine the effectiveness of the various peroxidase labelled sheep anti-rabbit Ig antibody or Fab fragment to detect intracellular IgG. Cell suspensions from the spleen and the lymph nodes of hyperimmunized rabbits were prepared, smeared on slides and fixed with a mixture of alcohol-ether following published procedures (Avrameas, 1969; Avrameas et al., 1971). Furthermore, the slides carrying the cells were fixed for 16 hr at +4°C with 4 per cent paraformaldehyde in 0.1 M cacodylate buffer pH 7-2. The slides were washed in buffered saline and then utilized for the detection of IgG on the same day and 24 and 48 hr later. Due to the continuous action of formaldehyde which proceeds even after the washing of the cellular preparations, the cells become progressively more extensively cross linked. The slides were incubated at room temperature for 1-3 hr with the peroxidase labelled preparations diluted to contain the same quantity of antibody (0.250 mg/ml), washed with saline and stained for peroxidase (Avrameas et al., 1971). To detect the antigen, the peroxidase labelled antibody or Fab fragment obtained by method 2, were stored for one week at 4°C, centrifuged (20,000 g, 20 min) and then employed; storage was necessary to completely eliminate a background stain which was observed if the conjugates were used at once. When the slides were fixed with the alcohol-ether mixture, essentially the same number of positive cells were counted with all the peroxidase labelled antibody or Fab preparations tested. However, the intensity of staining was higher with the preparations made by labelled antibody or Fab prepared with method 2. When slides fixed with formaldehyde were used immediately after washing, approximately the same number of positive cells were counted with all the peroxidase labelled preparations. To detect intracellular IgG, 1 hr of incuba-
Communications to the Editors
1179
tion was sufficient with the labelled antibody or Fab prepared by method 2, while 3 hr were needed with the labelled antibody prepared by the method 1. When sfides, fixed with formaldehyde and employed 24 hr later, were stained with antibody labelled by method 1, some positive cells were observed but the majority of the cells were stained only on the surface. The Fab labelled by method 1 or 2 gave the same numbers of positive ceils although the latter preparation gave the most intensively stained cells. When slides fixed with formaldehyde were employed 48 hr later, negative results were obtained with the antibody labelled by method 1. Many positive cells were observed with the Fab labelled by method 1 and with the antibody labelled by method 2. The best results were obtained with the Fab labelled by method 2 where well stained cells, equal in number with those counted in the cellular preparation fixed with the alcohol-ether mixture were found. The experiments described above have shown that either method 1 or method 2 is equally effective in detecting intracellular antigen when the conjugates can easily penetrate in the interior of the cell. Easy penetration is achieved with cells fixed with organic solvents, such as the alcohol-ether mixture, but not with cells fixed with cross-linking substances, such as formaldehyde. In the latter case, antibody or Fab labelled by the method described in this paper is noticeably more effective for the detection of antigen, the best results being obtained with Fab. Laboratoire Chimie des Protgines Institut de Recherches Scientifiques sur le Cancer B.P. No 8, VillejuiJ, (F-94) France
S. AVRAMEAS T. TERNYNCK
REFERENCES Avrameas S. (1969)Immunochemistry 6, 43. Avrameas S. (1970)Int. Rev. Cytol. 27, 349. Avrameas S. and Ternynck T. (1969)Immunochemistry 6, 53. Avrameas S., Taudou B. and Ternynck T. (1971)Int. Arch. Allergy 40, 161. Porter R. R. (1959)Biochem.J. 73, 119. Shapiro A. L., Vinuela E. and Maizel J. v., Jr. (1967)Biochem. biophys. Res. Commun 28, 815. UrielJ. (1966)Bull. Soc. chim. Biol. 48, 969.
C O M M U N I C A T I O N TO THE EDITORS Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration (Received It June 1971)
Enzyme labelled antibodies have been used cytochemically to detect antigens in tissues (Avrameas, 1970). With this technique, the most satisfactory results were obtained when peroxidase was employed as the enzyme label and glutaraldehyde was utilized to link the enzyme to the antibody (Avrameas, 1970, 1969). Although highly sensitive and capable of detecting small amounts of antigen, the immunoenzyme technique is limited by the penetration of the enzyme labelled antibody into the cell. This limitation is unimportant when organic fixatives like alcohol or acetone are used to fix the cells but it is particularly pronounced when cross-linking fixatives are used, like glutaraldehyde or formaldehyde, these being substances currently employed in electron microscopy. This paper presents a two step procedure which uses glutaraldehyde to prepare both peroxidase-antibody and peroxidase-Fab conjugates. The molar ratio of antibody or Fab to peroxidase is 1 : 1 and the conjugates can easily penetrate into the interior of well preserved formaldehyde fixed cells. The coupling of peroxidase to an antibody using a two step procedure via glutaraldehyde action is based on the observation that peroxidase alone could not be insolubilized even with an excess of glutaraldehyde (Avrameas and Ternynck, 1969). A possible explanation is that the free amino groups of the peroxidase molecule reacted with one of the two aldehyde groups of glutaraldehyde but that the configuration of the peroxidase derivative obtained was such that the second free aldehyde group was unable to combine with another peroxidase molecule. The free aldehyde group resting active should be available for combination with the amino groups of a protein added subsequently, providing that the two groups approach close enough. With this possibility in mind, we have tried adding antibody to the glutaraldehyde treated peroxidase under various experimental conditions. COUPLING OF GLUTARALDEHYDE TREATED PEROXIDASE WITH ANTIBODY OR Fab Sheep anti-rabbit IgG and anti-rat IgG antisera were prepared as described elsewhere (Avrameas et al., 1971). Sheep anti-rabbit Ig and anti-rat IgG antibody were isolated by passage of the whole antisera on an immunoadsorbent prepared by co-polymerization at pH 5 of 4 parts bovine serum albumin and one part IgG (Avrameas and Ternynck, 1969). The antibody preparations were subsequently filtered through Sephadex G-200 and the IgG containing fractions pooled for use. Fab-antibody fragments were prepared by the method of Porter (1959) using papain at a concentration of 2 per cent and a digestion o f 2 hr at 37°C. Fab and Fc fragments were separated from the non digested antibody by 1175
1176
Communications to the Editors
gel filtration of the reaction mixture (100 mg) through a Sephadex G-100 column (90 × 1.5 cm). The Fab fragment was then separated from the Fc component using a DEAE-cellulose column (20 × 2.5 cm) equilibrated with 0.0175 M phosphate buffer pH 6.9. Under these conditions, the Fab fragment is eluted in the breakthrough volume. Three different lots of horse radish peroxidase RZ3 were used, two from Boehringer, Mannheim (Germany) designated peroxidase PB1 and PB2 and one from Worthington, Freehold, New Jersey, designated peroxidase PW. To find the best conditions for the coupling of glutaraldehyde treated peroxidase with antibody, experiments were performed using buffers of several pH values and varying the concentrations of activated peroxidase and antibody. After a set of trials and taking into account the whole of the results obtained, the following technique was finally adopted. Ten mg of peroxidase were dissolved in 0.2 ml 0" 1 M phosphate buffer pH 6.8 containing 1.25 per cent glutaraldehyde (TAAB Lab., Reading, England). The solution was allowed to stand for 18 hr at room temperature and then filtered through a Sephadex G-25 column (60 × 0.9 cm) equilibrated with 0.15 M NaCI. The brown colored fractions containing the activated peroxidase were pooled and if needed concentrated to 1 ml with a Diaflo PM 10 membrane. To this solution, 1 ml of 0.15M NaC1 containing either 5 mg of antibody or 2.5 mg of Fab was added followed by the addition of 0.1 ml of 1 M carbonate-bicarbonate buffer pH 9.5. After 24 hr at 4°C, 0" 1 ml of 0"2 M solution of lysine was added and the mixture was then allowed to stand for an additional 2 hr. The preparation was then dialyzed at +4°C against several changes of buffered saline. The labelled antibody preparation was precipitated at +4°C with an equal volume of saturated neutral solution of ammonium sulfate. The precipitate was washed twice with half saturated ammonium sulfate solution, dissolved in a minimal volume of distilled water and exhaustively dialysed against buffered saline. The labelled Fab preparation was employed without treatment with ammonium sulfate. Both antibody or Fab peroxidase labelled preparations were centrifuged (20,1)00 g, 20 min) and stored at +4°C for 3 months without noting any loss in their capacity to detect the antigen. When a high proportion of peroxidase labelled antibody or Fab is wanted, as in the case of electron microscopic studies, 15 mg of peroxidase instead of 10 mg should be used. If wanted, peroxidase labelled antibody or Fab can be separated from free antibody or Fab by filtration of the preparation on a sephadex G-200 column (100 × 2 cm), although such treatment does not add any practical advantage for the detection of antigen. CHARACTERIZATION OF THE COMPOUNDS Chromatography of the three different lots of peroxidase on Sephadex G-200 column (100X2cm) calibrated for molecular weight determination gave a single peak corresponding to a molecular weight of 40,000. Chromatography of glutaraldehyde treated peroxidase PB1 and PB2 gave two peaks corresponding to molecular weights of 80,000 and 40,000, the former peak accounting for 23 per cent of the PB1 preparation and for 10 per cent of the PB2 preparation. The treated PW preparation gave a single chromatographic peak corresponding to a molecular weight of 40,000. Similar results were obtained when the three
Communications to the Editors
1 177
glutaraldehyde treated peroxidase preparations were run in sodium dodecyl sulfate (SDS 1%)-acrylamide (5%)/agarose (0"8%) gel electrophoresis (Shapiro et al., 1967; Uriel, 1966), and also when they were subjected to analytical ultracentrifugation. It thus appears that the treatment of peroxidase with glutaraldehyde produces a relatively low proportion of peroxidase dimers. When a mixture of antibody and peroxidase was applied to the above Sephadex G-200 column, two distinct chromatographic peaks were obtained; readings at 403 m/z revealing the chromatographic profile of peroxidase (Fig. 1A). When the peroxidase labelled antibody, prepared by the one step method previously described (Avrameas, 1969) and in the following termed method 1, was chromatographed only one asymmetric peak was obtained which emerged with the void volume of the column (Fig. 1B). With the peroxidase labelled antibody prepared by the present two step method, in the following termed method 2, also only one asymmetric peak was obtained (Fig. 1C). The readings at 403 m/z showed I 0.150
01
i
i i i i i i
0,100
0.050
i
0.150
® I~ 0.100
d 0.050
i
OJSO
© 0,100
0.050
20
40 60 TUBE NUMBERS
80
Fig. l. Chromatography on Sephadex G-200 column (100x 2 cm) equilibrated with 0-1 M phosphate buffer p H 6.8 o f (A): a mixture of sheep antibody anti-rabbit Ig and peroxidase: (B): peroxidase labelled sheep antibody anti-rabbit Ig prepared by the one step procedure; (C): peroxidase labelled sheep antibody anti-rabbit prepared by the two step procedure. ( ) optical density at 280 m/~ (. . . . . ) optical density at 403 m/z.
1178
Communications to the Editors
peroxidase eluted just before the position of IgG and corresponding to a molecular weight higher or equal to 200,000 while the reading at 280 m g showed a maximum in the position of IgG (Fig. 1C). The peroxidase labelled antibody preparations obtained by method 1 and method 2 were subjected to SDS-acrylamide/agarose gel electrophoresis. The method 1 labelled antibody hardly penetrated the gel. With the method 2 labelled antibody, two main bands were noted; one had a molecular weight of approximately 170,000 corresponding to free antibody and the other to a molecular weight of 210,000 corresponding to antibody-peroxidase conjugates; the latter was stained using the specific substrate for peroxidase. Similar experiments were carried out with peroxidase labelled Fab prepared by methods 1 and 2. Coupling by method 2 gave rise to two main fractions. The first has a molecular weight of 80,000 and the second of 40,000. In the 80,000 molecular weight fraction, Fab-peroxidase conjugates were found and, when peroxidase PB1 or PB2 were used, dimers of peroxidase molecules. In the 40,000 molecular weight fraction, free peroxidase was found and, when the quantity of peroxidase employed for the coupling was low, free Fab. Coupling of peroxidase to Fab by method 1 gave rise to a highly heterogeneous population of conjugates having a molecular weight range from 80,000 to 160,000. EFFECTIVENESS OF THE CONJUGATES TO DETECT INTRACELLULAR ANTIGEN Experiments were carried out to determine the effectiveness of the various peroxidase labelled sheep anti-rabbit Ig antibody or Fab fragment to detect intracellular IgG. Cell suspensions from the spleen and the lymph nodes of hyperimmunized rabbits were prepared, smeared on slides and fixed with a mixture of alcohol-ether following published procedures (Avrameas, 1969; Avrameas et al., 1971). Furthermore, the slides carrying the cells were fixed for 16 hr at +4°C with 4 per cent paraformaldehyde in 0.1 M cacodylate buffer pH 7-2. The slides were washed in buffered saline and then utilized for the detection of IgG on the same day and 24 and 48 hr later. Due to the continuous action of formaldehyde which proceeds even after the washing of the cellular preparations, the cells become progressively more extensively cross linked. The slides were incubated at room temperature for 1-3 hr with the peroxidase labelled preparations diluted to contain the same quantity of antibody (0.250 mg/ml), washed with saline and stained for peroxidase (Avrameas et al., 1971). To detect the antigen, the peroxidase labelled antibody or Fab fragment obtained by method 2, were stored for one week at 4°C, centrifuged (20,000 g, 20 min) and then employed; storage was necessary to completely eliminate a background stain which was observed if the conjugates were used at once. When the slides were fixed with the alcohol-ether mixture, essentially the same number of positive cells were counted with all the peroxidase labelled antibody or Fab preparations tested. However, the intensity of staining was higher with the preparations made by labelled antibody or Fab prepared with method 2. When slides fixed with formaldehyde were used immediately after washing, approximately the same number of positive cells were counted with all the peroxidase labelled preparations. To detect intracellular IgG, 1 hr of incuba-
Communications to the Editors
1179
tion was sufficient with the labelled antibody or Fab prepared by method 2, while 3 hr were needed with the labelled antibody prepared by the method 1. When sfides, fixed with formaldehyde and employed 24 hr later, were stained with antibody labelled by method 1, some positive cells were observed but the majority of the cells were stained only on the surface. The Fab labelled by method 1 or 2 gave the same numbers of positive ceils although the latter preparation gave the most intensively stained cells. When slides fixed with formaldehyde were employed 48 hr later, negative results were obtained with the antibody labelled by method 1. Many positive cells were observed with the Fab labelled by method 1 and with the antibody labelled by method 2. The best results were obtained with the Fab labelled by method 2 where well stained cells, equal in number with those counted in the cellular preparation fixed with the alcohol-ether mixture were found. The experiments described above have shown that either method 1 or method 2 is equally effective in detecting intracellular antigen when the conjugates can easily penetrate in the interior of the cell. Easy penetration is achieved with cells fixed with organic solvents, such as the alcohol-ether mixture, but not with cells fixed with cross-linking substances, such as formaldehyde. In the latter case, antibody or Fab labelled by the method described in this paper is noticeably more effective for the detection of antigen, the best results being obtained with Fab. Laboratoire Chimie des Protgines Institut de Recherches Scientifiques sur le Cancer B.P. No 8, VillejuiJ, (F-94) France
S. AVRAMEAS T. TERNYNCK
REFERENCES Avrameas S. (1969)Immunochemistry 6, 43. Avrameas S. (1970)Int. Rev. Cytol. 27, 349. Avrameas S. and Ternynck T. (1969)Immunochemistry 6, 53. Avrameas S., Taudou B. and Ternynck T. (1971)Int. Arch. Allergy 40, 161. Porter R. R. (1959)Biochem.J. 73, 119. Shapiro A. L., Vinuela E. and Maizel J. v., Jr. (1967)Biochem. biophys. Res. Commun 28, 815. UrielJ. (1966)Bull. Soc. chim. Biol. 48, 969.