68
BIOTIN-BINDING PROTEINS
[6]
[6] N o n g l y c o s y l a t e d A v i d i n
By YAFFA HILLER, EDWARDA. BAYER, and MEIR WILCHEK We have recently found ~ that a commercial preparation of egg-white avidin2 contains about 30% of a nonglycosylated form of the tetrameric protein. During the course of studies on this protein, we developed a method for the isolation of the nonglycosylated tetramer based on the removal of glycosylated forms 3 using appropriate lectin columns. Since avidin is known to contain both mannose and N-acetylglucosamine residues, 4 affinity columns containing concanavalin A (Con A) or wheat germ agglutinin can be used for this purpose. Con A is the lectin of choice because it is relatively inexpensive and because immobilized forms are obtainable from a variety of commercial sources. Even though no method is currently available for the experimental deglycosylation of avidin, 5 we describe here a method for the separation and isolation of the fully nonglycosylated tetramer from mixtures of glycosylated and partially nonglycosylated forms of the protein. This method will undoubtedly be useful when deglycosylation procedures are eventually described. 6
J Y. Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). z Batch 1283, Belovo Soc. Coop. (Bastogne, Belgium). 3 Glycosylated forms of avidin refer to tetramers containing either fully glycosylated monomers, monomers bearing truncated oligosaccharides, or mixtures of glycosylated and nonglycosylated monomers. 4 R. J. DeLange and T.-S. Huang, J. Biol. Chem. 246, 698 (1971). 5 In this chapter, a distinction is made between "nonglycosylation" and "deglycosylation"; in the former case the mechanism by which egg-white avidin has "lost" its oligosaccharide chains is unknown, wheras in the latter case the oligosaccharide residues of glycosylated forms of avidin are removed experimentally. Preliminary results using endoglycosidase F gave promising results in the deglycosylation of certain commercial preparations of avidin (e.g., Belovo) but not with others (e.g., avidin Ex, STC Laboratories Inc., Winnipeg, MB), suggesting qualitative differences in the oligosaccharide content of the two products (T. Viswanatha, personal communication). 6 In our laboratory, we have isolated from nature bacterial strains that are capable of producing deglycosylated avidin. We are currently in the process of isolating the enzyme(s) responsible for this activity.
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[6]
NONGLYCOSYLATEDAVIDIN II
Applied
~(H2o)
"~ E
I
I
i
I
Buffer
i
69 [
I
I
I
aMM
1
o 2.c E 0
Cl
BIOTIN-BINDING PROTEINS
[6]
[6] N o n g l y c o s y l a t e d A v i d i n
By YAFFA HILLER, EDWARDA. BAYER, and MEIR WILCHEK We have recently found ~ that a commercial preparation of egg-white avidin2 contains about 30% of a nonglycosylated form of the tetrameric protein. During the course of studies on this protein, we developed a method for the isolation of the nonglycosylated tetramer based on the removal of glycosylated forms 3 using appropriate lectin columns. Since avidin is known to contain both mannose and N-acetylglucosamine residues, 4 affinity columns containing concanavalin A (Con A) or wheat germ agglutinin can be used for this purpose. Con A is the lectin of choice because it is relatively inexpensive and because immobilized forms are obtainable from a variety of commercial sources. Even though no method is currently available for the experimental deglycosylation of avidin, 5 we describe here a method for the separation and isolation of the fully nonglycosylated tetramer from mixtures of glycosylated and partially nonglycosylated forms of the protein. This method will undoubtedly be useful when deglycosylation procedures are eventually described. 6
J Y. Hiller, J. M. Gershoni, E. A. Bayer, and M. Wilchek, Biochem. J. 248, 167 (1987). z Batch 1283, Belovo Soc. Coop. (Bastogne, Belgium). 3 Glycosylated forms of avidin refer to tetramers containing either fully glycosylated monomers, monomers bearing truncated oligosaccharides, or mixtures of glycosylated and nonglycosylated monomers. 4 R. J. DeLange and T.-S. Huang, J. Biol. Chem. 246, 698 (1971). 5 In this chapter, a distinction is made between "nonglycosylation" and "deglycosylation"; in the former case the mechanism by which egg-white avidin has "lost" its oligosaccharide chains is unknown, wheras in the latter case the oligosaccharide residues of glycosylated forms of avidin are removed experimentally. Preliminary results using endoglycosidase F gave promising results in the deglycosylation of certain commercial preparations of avidin (e.g., Belovo) but not with others (e.g., avidin Ex, STC Laboratories Inc., Winnipeg, MB), suggesting qualitative differences in the oligosaccharide content of the two products (T. Viswanatha, personal communication). 6 In our laboratory, we have isolated from nature bacterial strains that are capable of producing deglycosylated avidin. We are currently in the process of isolating the enzyme(s) responsible for this activity.
METHODS IN ENZYMOLOGY, VOL. 184
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
[6]
NONGLYCOSYLATEDAVIDIN II
Applied
~(H2o)
"~ E
I
I
i
I
Buffer
i
69 [
I
I
I
aMM
1
o 2.c E 0
Cl
