Electrophoresis 1990,II. 505-506
Isoelectric focusing of biotinylated lectins
505
Short communications Hiroshi Harada' Maki Kondo' Takashi Yamaguchi2 'Honen Company, Tokyo 2SugiyamaChemical and Industrial Laboratory, Yokohama
Estimation of biotinylated lectin by isoelectric focusing Concanavalin A (Con A) was biotinylated to various degrees using N-biotinyl-waminocaproic-acid-N-hydroxysuccinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3-9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that ofthe original ConA by 2.2 k 0.6 had the strongest binding activity for ovalbumin.
Lectins provide a powerful means for studying carbohydrates and their derivatives, both in solution and on cell surfaces. They are widely used for preparative and analytical purposes in biochemistry, cell biology, immunology and related areas, particularly with glycoconjugates. When working with lectins, biotinylated lectins are generally used to investigate the architecture of cell surfaces and changes resulting from malignant transformation. This is because biotinylated lectins can be visualized with great sensitivity by the derivatives of avidin [ 11. Lectins can easily be biotinylated by different types of commercially available biotinylation reagents. However, the extent of biotinylation on lectin cannot be determined by colorimetry using dimethylaminocinnamaldehyde [21. Although this is the general assay method for the quantitative analysis of biotin, the protein portion of biotinylated lectins inhibits colorimetric reactions. In assessing the binding capacity of biotinylated lectins toward glycoprotein, the extent of biotinylation on lectins was easy to estimate by isoelectric focusing (IEF). Also, there was an optimal biotinylation reagent and lectin ratio for the biotinylation of lectins without loss of binding activity toward glycoprotein, as will be discussed below.
Figure 1 . IEF of biotinylated ConA in PhastGel IEF 3-9. Samples (1 pL) were applied with the sample applicator and IEF conducted according to the method of Olsson el al. [61. The protein patterns were visualized by Coomassie Brilliant Blue staining 161. A Pharmacia Calibration Kit 3-10 was used as the p l marker. Samples: (1) ConA, (2) BC8, (3) BC 16, (4) BC24, (5)BC32 and ( 6 ) BC40.
To obtain concanavalin A (ConA) biotinylated to various degrees, N-biotinyl-w-aminocaproic-acid-N-hydroxy succinimide ester [31 was used as biotinylation reagent. Fifteen mg of ConA (obtained from Honen Co.) were dissolved in 5 m L Colorimetry using dimethylaminocinnamaldehyde (21 is genof 0.1 M sodium carbonate buffer (containing 0.1 M glucose, erally used to determine biotin content. However, in the case of pH 9.0) and 4.25 mg of N-biotinyl-w-aminocaproic-acid-N-biotin attached to protein, this method is not suitable for this hydroxy succinimide ester, in 1 mL of dimethyl sulfoxide. The determination since the protein portion of biotinylated probiotinylation reagent solution (8,16,24,32 and40 pL) was ad- teins inhibits colorimetric reactions of dimethylaminocinnaded to each 1 mL of ConA solution, followed by incubation for maldehyde. Thus, the amount of biotin attached to ConA was 2 h at room temperature. Biotinyl-reaction solutions were dialyzed against H 2 0 , followed by lyophilization. The bio- determined by the dye-binding method using 4-hydroxyazobenzene-2-carboxylic acid (HABA) [41. With the molecular tinylated ConAs thus obtained were desiganted BC8, BC 16, weight of ConA estimated to be 100 000, the average amounts BC24, BC32 and BC40 according to the volume of biotinylof biotin per BC8, BC 16, BC24, BC32 and BC40 were calcuation reagent solution (8, 16,24,32 and 40 pL, respectively). ConA, BC8, BC 16, BC24, BC32 and BC40 were dissolved in lated to be 1.5,2.5,6.3,9.2,9.7 biotinmolecules per ConA molecule, respectively. From these values, it is evident that theexH,O, each at a concentration of 1 mg/mL, and then analyzed tent of biotinylation on ConA increases with the volume of by IEF using PhastGel IEF 3-9. It is evident (Fig. I ) that the isoelectric point (pl) of biotinylated ConA decreases with in- biotinylation reagent solution used. Thus, the decrease in the p l value of biotinylated ConA corresponds to the increase in creasing volume of biotinylation reagent. the amount of biotin per ConA molecule. The above biotinylated ConAs were analyzed by dot blotting forbinding tostreptoavidin [51. BC8,BC16,BC24,BC32and Correspondence: Dr. Hiroshi Harada, Honen Co., 1-2-3 Ohtemachi, BC40in 15 mMphosphatebuffered saline, pH 7.4, were placed Chiyoda-ku, Tokyo 100, Japan in each row on a nitrocellulose membrane using Bio-Dot apparatus (Bio-Rad Laboratories, Tokyo). The membrane was Abbreviation: ConA, concanavalin A; HABA, 4-hydroxybenzene-2'-carthen washed for 40 min at room temperature in Tris-Tweenboxylic acid; HRP, horseradish peroxidase; IEF, isoelectric focusing; pZ, saline (10 mM Tris-HC1, pH 7 . 5 , 0 . 0 5 o/o Tween 20, 0.15 M isoelectric point 0VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
0173-0835/90/0606-0505 $02.5010
506
Electrophoresis 1990, I I , 505-506
H. Harada el al.
Figure 2. Dot blotting of biotinylated ConA. BC:8(a), BC 16(b), BC24(c), BC32(d) and BC40(e) in 15 mM phosphate-buffxed saline, pH 7.4, were placed in each row on a nitrocellulose membrane using the Bio-Dot apparatus: (1) 400 ng, (2) 200 ng, (3) 100 ng, (4) 50 ng, (5) 25 ng, (6) 12.5 ng, (7) 6.2 ng and (8) 3.1 ng. After the biotinylated ConAs had reacted with HRP-streptoavidin, the membrane was washed iin Tris-Tween-saline and then allowed to react with 3,3’-diaminobenzidine. For details see text.
NaCl) with several changes. After the biotinylated ConAs on the nitrocellulose had reacted for 15 min at room temperature with 30 mL of horseradish peroxidase (HRP)-streptoavidin HRP-streptoavidin solution from BioCienex (San Ramon, CA) was diluted with 50 parts of Tris-Tween-saline - the membrane was washed for 40 min at room temperature in several changes of Tris-Tween-saline and then allowed to react with 3,3’-diaminobenzidine (0.03 % in 15 mM phosphate-buffered saline, pH 7.4, containing 0.003 % H,O,) at room temperature until adequate color development, usually 3-5 min. BC 16, BC24, BC32 and BC40 (Fig 2, b throughe) at 12.5 to 400 ng levels are capable of developing dots, thus showing biotinylated ConAs to have the same binding activity toward streptoavidin. This indicates that biotinylated ConAs with higher degrees of biotinylation do not have significantly stronger binding activity toward strepto,avidin. BC8, BC 16, BC24, BC32 and BC40 were examined for their binding activity toward glycoprotein (using ovalbumin) by dot blotting [SI. Ovalbumin in 15 mM phosphate buffered saline, pH 7.4, was placed in each row on a nitrocellulose membrane using the Bio-Dot apparatus. The membrane was washed for 40 min at room temperature in several changes ofTris-Tweensaline. The membrane was cut into 6 strips, each of which was then reacted withBC8,BC16, BC24, BC32 andBC40inTrisTween-saline at a concentration of 2 bg/mL for 60 rnin at room temperature followed by dot development as described above using HRP-streptoavidin and 3J’-diaminobenzidine for ovalbumin detection. It is generally known that biotin molecules can be coupled to either antigens or antibodies, usually without loss of biological activity of the latter. However, Fig. 3 (a through e) shows a critical difference in detection sensitivity for ovalbumin amon,g biotinylated ConAs. There is thus an optimal biotinylatioln reagent and lectin ratio for biotin-coupling reactions without loss of binding activity of lectin toward glycoprotein. BC 116 having a p1 value
Figure 3. Interaction of ovalbumin and biotinylated ConA. Ovalbumin in 15 mM phosphate-buffered saline, pH 7.4, was placed in each row on a nitrocellulosemembrane usingthe Bio-Dot apparatus:(1)7.4 pg,(2)2.5 pg, (3) 823 ng, (4) 274 ng, (5) 9 1.4 ng, (6) 30 ng, (7) 10 ng, (8) 3.4 ng and (9) 1.1 ng. The membrane wascut into6 strips,eachofwhichwasthenreactedwith BC8(a), BC 16(b), BC24(c), BC 32(d) and BC4O(e)in Tris-Tween-saline at a concentration of 2 pg/mL for 60 rnin at room temperature. The dot development procedure with HRP-streptoavidin and 3,3’-diaminobenzidine was the same as in Fig. 2.
lower than that ofthe original ConA by 2.2 k0.6 was found to have the greatest binding activity toward ovalbumin. Thus, the recommended N-biotinyl-o-aminocaproic-acid-N-hydroxy succinimide ester and ConA ratio for biotinylation of ConA is 1O:l mol/mol. Further, we used IEF to estimate the degree of biotinylation on other lectins. Biotinylated peanut agglutinin, soybean agglutinin and lentil lectin, all differing in biotin content, were prepared and analyzed by IEF. The p l o f biotinylated lectins was found to decrease with increasing volume of biotinylation reagent (datanot shown). Thus, I E F is preferabel to the HABA method for estimating the extent of biotinylation on biotinylated lectins for the following reasons: (i) I E F can be carried out in less than 2 h and (ii) expensive reagents such as avidin, required in the HABA method, are not necessary. I E F can also be ustd to estimate the extent of biotinylation not only onlectins but also on immunoglobulin. Received October 9, 1989; in revised form January 19, 1990
References [ 11 Bayer, E. A., Skutelsky, E. and Wilchek, M., Method Enzymol. 1982,
83, 195-215. [21 McCormick,D. B. andRoth,J. A.,Anal. Biochem. 1970,34,226-236. [31 Finn, F. M., Titus, G. and Hofman, K., Biochemistry 1984, 23, 25 54-25 5 8. 141 Green, N. M., Biochem. J. 1965,94, 23c. 151 Harada, H. and Koyama, F., Physico-Chem. Biol. (Seibutsubutsurikagaku) 1987,3I, 165-168. 161 Olsson, I., Axio-Fredriksson, U.-B., Degerman, M. and Olsson, B., Electrophoresis 1988,9, 16-22.
Isoelectric focusing of biotinylated lectins
505
Short communications Hiroshi Harada' Maki Kondo' Takashi Yamaguchi2 'Honen Company, Tokyo 2SugiyamaChemical and Industrial Laboratory, Yokohama
Estimation of biotinylated lectin by isoelectric focusing Concanavalin A (Con A) was biotinylated to various degrees using N-biotinyl-waminocaproic-acid-N-hydroxysuccinimide ester as the biotinylation reagent, and then analyzed by isoelectric focusing using PhastGel IEF 3-9. The isoelectric points of biotinylated ConAs were found to decrease with increasing concentration of the biotinylation reagent. Analysis by isoelectric focusing followed by dot blotting clearly indicated that the biotinylated ConA with an isoelectric point lower than that ofthe original ConA by 2.2 k 0.6 had the strongest binding activity for ovalbumin.
Lectins provide a powerful means for studying carbohydrates and their derivatives, both in solution and on cell surfaces. They are widely used for preparative and analytical purposes in biochemistry, cell biology, immunology and related areas, particularly with glycoconjugates. When working with lectins, biotinylated lectins are generally used to investigate the architecture of cell surfaces and changes resulting from malignant transformation. This is because biotinylated lectins can be visualized with great sensitivity by the derivatives of avidin [ 11. Lectins can easily be biotinylated by different types of commercially available biotinylation reagents. However, the extent of biotinylation on lectin cannot be determined by colorimetry using dimethylaminocinnamaldehyde [21. Although this is the general assay method for the quantitative analysis of biotin, the protein portion of biotinylated lectins inhibits colorimetric reactions. In assessing the binding capacity of biotinylated lectins toward glycoprotein, the extent of biotinylation on lectins was easy to estimate by isoelectric focusing (IEF). Also, there was an optimal biotinylation reagent and lectin ratio for the biotinylation of lectins without loss of binding activity toward glycoprotein, as will be discussed below.
Figure 1 . IEF of biotinylated ConA in PhastGel IEF 3-9. Samples (1 pL) were applied with the sample applicator and IEF conducted according to the method of Olsson el al. [61. The protein patterns were visualized by Coomassie Brilliant Blue staining 161. A Pharmacia Calibration Kit 3-10 was used as the p l marker. Samples: (1) ConA, (2) BC8, (3) BC 16, (4) BC24, (5)BC32 and ( 6 ) BC40.
To obtain concanavalin A (ConA) biotinylated to various degrees, N-biotinyl-w-aminocaproic-acid-N-hydroxy succinimide ester [31 was used as biotinylation reagent. Fifteen mg of ConA (obtained from Honen Co.) were dissolved in 5 m L Colorimetry using dimethylaminocinnamaldehyde (21 is genof 0.1 M sodium carbonate buffer (containing 0.1 M glucose, erally used to determine biotin content. However, in the case of pH 9.0) and 4.25 mg of N-biotinyl-w-aminocaproic-acid-N-biotin attached to protein, this method is not suitable for this hydroxy succinimide ester, in 1 mL of dimethyl sulfoxide. The determination since the protein portion of biotinylated probiotinylation reagent solution (8,16,24,32 and40 pL) was ad- teins inhibits colorimetric reactions of dimethylaminocinnaded to each 1 mL of ConA solution, followed by incubation for maldehyde. Thus, the amount of biotin attached to ConA was 2 h at room temperature. Biotinyl-reaction solutions were dialyzed against H 2 0 , followed by lyophilization. The bio- determined by the dye-binding method using 4-hydroxyazobenzene-2-carboxylic acid (HABA) [41. With the molecular tinylated ConAs thus obtained were desiganted BC8, BC 16, weight of ConA estimated to be 100 000, the average amounts BC24, BC32 and BC40 according to the volume of biotinylof biotin per BC8, BC 16, BC24, BC32 and BC40 were calcuation reagent solution (8, 16,24,32 and 40 pL, respectively). ConA, BC8, BC 16, BC24, BC32 and BC40 were dissolved in lated to be 1.5,2.5,6.3,9.2,9.7 biotinmolecules per ConA molecule, respectively. From these values, it is evident that theexH,O, each at a concentration of 1 mg/mL, and then analyzed tent of biotinylation on ConA increases with the volume of by IEF using PhastGel IEF 3-9. It is evident (Fig. I ) that the isoelectric point (pl) of biotinylated ConA decreases with in- biotinylation reagent solution used. Thus, the decrease in the p l value of biotinylated ConA corresponds to the increase in creasing volume of biotinylation reagent. the amount of biotin per ConA molecule. The above biotinylated ConAs were analyzed by dot blotting forbinding tostreptoavidin [51. BC8,BC16,BC24,BC32and Correspondence: Dr. Hiroshi Harada, Honen Co., 1-2-3 Ohtemachi, BC40in 15 mMphosphatebuffered saline, pH 7.4, were placed Chiyoda-ku, Tokyo 100, Japan in each row on a nitrocellulose membrane using Bio-Dot apparatus (Bio-Rad Laboratories, Tokyo). The membrane was Abbreviation: ConA, concanavalin A; HABA, 4-hydroxybenzene-2'-carthen washed for 40 min at room temperature in Tris-Tweenboxylic acid; HRP, horseradish peroxidase; IEF, isoelectric focusing; pZ, saline (10 mM Tris-HC1, pH 7 . 5 , 0 . 0 5 o/o Tween 20, 0.15 M isoelectric point 0VCH Verlagsgesellschaft mbH D-6940 Weinheim, 1990
0173-0835/90/0606-0505 $02.5010
506
Electrophoresis 1990, I I , 505-506
H. Harada el al.
Figure 2. Dot blotting of biotinylated ConA. BC:8(a), BC 16(b), BC24(c), BC32(d) and BC40(e) in 15 mM phosphate-buffxed saline, pH 7.4, were placed in each row on a nitrocellulose membrane using the Bio-Dot apparatus: (1) 400 ng, (2) 200 ng, (3) 100 ng, (4) 50 ng, (5) 25 ng, (6) 12.5 ng, (7) 6.2 ng and (8) 3.1 ng. After the biotinylated ConAs had reacted with HRP-streptoavidin, the membrane was washed iin Tris-Tween-saline and then allowed to react with 3,3’-diaminobenzidine. For details see text.
NaCl) with several changes. After the biotinylated ConAs on the nitrocellulose had reacted for 15 min at room temperature with 30 mL of horseradish peroxidase (HRP)-streptoavidin HRP-streptoavidin solution from BioCienex (San Ramon, CA) was diluted with 50 parts of Tris-Tween-saline - the membrane was washed for 40 min at room temperature in several changes of Tris-Tween-saline and then allowed to react with 3,3’-diaminobenzidine (0.03 % in 15 mM phosphate-buffered saline, pH 7.4, containing 0.003 % H,O,) at room temperature until adequate color development, usually 3-5 min. BC 16, BC24, BC32 and BC40 (Fig 2, b throughe) at 12.5 to 400 ng levels are capable of developing dots, thus showing biotinylated ConAs to have the same binding activity toward streptoavidin. This indicates that biotinylated ConAs with higher degrees of biotinylation do not have significantly stronger binding activity toward strepto,avidin. BC8, BC 16, BC24, BC32 and BC40 were examined for their binding activity toward glycoprotein (using ovalbumin) by dot blotting [SI. Ovalbumin in 15 mM phosphate buffered saline, pH 7.4, was placed in each row on a nitrocellulose membrane using the Bio-Dot apparatus. The membrane was washed for 40 min at room temperature in several changes ofTris-Tweensaline. The membrane was cut into 6 strips, each of which was then reacted withBC8,BC16, BC24, BC32 andBC40inTrisTween-saline at a concentration of 2 bg/mL for 60 rnin at room temperature followed by dot development as described above using HRP-streptoavidin and 3J’-diaminobenzidine for ovalbumin detection. It is generally known that biotin molecules can be coupled to either antigens or antibodies, usually without loss of biological activity of the latter. However, Fig. 3 (a through e) shows a critical difference in detection sensitivity for ovalbumin amon,g biotinylated ConAs. There is thus an optimal biotinylatioln reagent and lectin ratio for biotin-coupling reactions without loss of binding activity of lectin toward glycoprotein. BC 116 having a p1 value
Figure 3. Interaction of ovalbumin and biotinylated ConA. Ovalbumin in 15 mM phosphate-buffered saline, pH 7.4, was placed in each row on a nitrocellulosemembrane usingthe Bio-Dot apparatus:(1)7.4 pg,(2)2.5 pg, (3) 823 ng, (4) 274 ng, (5) 9 1.4 ng, (6) 30 ng, (7) 10 ng, (8) 3.4 ng and (9) 1.1 ng. The membrane wascut into6 strips,eachofwhichwasthenreactedwith BC8(a), BC 16(b), BC24(c), BC 32(d) and BC4O(e)in Tris-Tween-saline at a concentration of 2 pg/mL for 60 rnin at room temperature. The dot development procedure with HRP-streptoavidin and 3,3’-diaminobenzidine was the same as in Fig. 2.
lower than that ofthe original ConA by 2.2 k0.6 was found to have the greatest binding activity toward ovalbumin. Thus, the recommended N-biotinyl-o-aminocaproic-acid-N-hydroxy succinimide ester and ConA ratio for biotinylation of ConA is 1O:l mol/mol. Further, we used IEF to estimate the degree of biotinylation on other lectins. Biotinylated peanut agglutinin, soybean agglutinin and lentil lectin, all differing in biotin content, were prepared and analyzed by IEF. The p l o f biotinylated lectins was found to decrease with increasing volume of biotinylation reagent (datanot shown). Thus, I E F is preferabel to the HABA method for estimating the extent of biotinylation on biotinylated lectins for the following reasons: (i) I E F can be carried out in less than 2 h and (ii) expensive reagents such as avidin, required in the HABA method, are not necessary. I E F can also be ustd to estimate the extent of biotinylation not only onlectins but also on immunoglobulin. Received October 9, 1989; in revised form January 19, 1990
References [ 11 Bayer, E. A., Skutelsky, E. and Wilchek, M., Method Enzymol. 1982,
83, 195-215. [21 McCormick,D. B. andRoth,J. A.,Anal. Biochem. 1970,34,226-236. [31 Finn, F. M., Titus, G. and Hofman, K., Biochemistry 1984, 23, 25 54-25 5 8. 141 Green, N. M., Biochem. J. 1965,94, 23c. 151 Harada, H. and Koyama, F., Physico-Chem. Biol. (Seibutsubutsurikagaku) 1987,3I, 165-168. 161 Olsson, I., Axio-Fredriksson, U.-B., Degerman, M. and Olsson, B., Electrophoresis 1988,9, 16-22.
