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The purification of avidin and its derivatives on 2-iminobiotin-6-aminohexyl-Sepharose 4B
The
Purification
of Avidin
and
Its Derivatives
2-lminobiotin-6-aminohexyl-Sepharose GAYLE
HENEY
Received
and
The pH-dependent avidin has been
interaction used in the
its fluorescent and iodinated of 2-iminobiotin-6-aminohexylSepharose ically eluted from the column conditions, i.e., 6 M guanidineeHC1. biotin
AND
0003-2697/8
A.
16.
derivatives. at pH
Avidin and 48 at pH 4. This affinity pH 1.5. required
ORR’
I980
between the cyclic guanidino design of an efficient affinity
analog isolation
its derivatives values between
of biotin. system
2.iminobiotin. for avidin
and
are retained by a column 9 and I I and are spectf-
isolation procedure to dissociate avidin
overcomes the harsh from an immobilkd
column.
The avidin biotin complex is characterized by an extremely low dissociation constant (Kn) of approximately lo-15 M (1). The tightness of this interaction has formed the basis for the useof the complex in several cytochemical techniques in membrane and molecular biology (2). For example, avidin biotin complex formation has been used successfully for the visualization and quantitication of receptors for lectin (3) polypeptide hormones (4). and other molecules (5,6) on the cell surface. Fluorescent and electrondenseavidin derivatives have been employed to observe biological membranes labeled with biotin (7) and for the ultrastructural localization of biotinylated intracellular components (8). Davidson’s group has described methods for electron microscopic gene mapping using specfic RNA biotin cytochrome c and avidin -ferritin complexes to visualize RNA -DNA hybridization (9). In many of these studies it would be desirable if an affinity isolation system were available for the isolation of biologically active avidin derivatives uncontaminated by damaged or unconjugated avidin molecules. Although the essentially covalent character of the nvidin biotin complex makes it an ideal choice for many of the above mentioned studies, it severely restricts its use for ’ To whom
46
GEORGE
December
on
all
correspondence
should
I /090092-05$02.00/0
Copyrnght c INI by Acddemx Prcsr. Inc. All right, of repruducuon ,n any form reserved
be addressed.
the affinity isolation of (a) avidin and its derivatives and (b) biotinylated components. Cuatrecasas and Wilchek have reported the isolation of avidin on a biocytin (biotin-t-IVlysyl)Sepharose 4B column ( IO). The conditions required for the elution of the specifically bound protein were a combination of low pH (1.5) and 6 M guanidine HCI: either alone did not effect elution. Although avidin is not inactivated by these harsh elution conditions, it is uncertain whether certain avidin derivatives. e.g., ferritin-conjugated avidin, would retain their desired biological activity. We have overcome this problem by replacing biotin with its cyclic guanidino analog. 2-iminobiotin. At high pH (>9) 2-iminobiotin exists primarily in its free base form and retains the tight specific binding to avidin characteristic of biotin ( I I. 17). At low pH, where it exists primarily in the salt form. it interacts weakly with avidin. This paper describes the synthesis of 7-iminobiotin-6-aminohexyl--Sepharose 4B and its use for the affinity isolation of avidin and of its biologically active fluorescent and iodinated derivatives. MATERIALS
AND METHODS
d-Biotin and avidin were obtained from Sigma Chemical Company, St. Louis, Mis-
AVIDIN
PURIFICATION
ON
IMMOBILIZED
souri. ci-[~arho,!l,/-“C]Biotin (56 mCi/ mmol) and “‘I-labeled Bolton Hunter reagent (1500 Ci/mmol) were from Amersham. Rhodamine B isothiocyanate was purchased fron! ICN. The cyclic guanidino analog of biotin, _‘-iminobiotin. was synthesized from the diamino carboxylic acid derivative of biotin, S-( 3,4-diamino-thiophan2-yl) pentanoic acid by reaction with cyanogen bromide (13,14).
,7-lminobiotin-6-aminohexyl-Sepharose di3. 2-lminohiotin (400 mg. 1.23 mmol) was added to 100 ml of 6-aminohexyl Sepharose 4B (40 ml of packed resin in H,O). prepared by the method of Porath ( 15). and the pH was adjusted to 4.8 with HBr ( I%, v/v). 1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (4.24 g, 10 mmol) was added portionwise over a period of IO min. The pH was kept at 4.8 throughout the reaction by the addition of 1% H Br and was constant after 3 to 5 h. The resin was washed with I M NaCl (2 liters), H,O (2 liters) and packed into a column. The binding capacity of the affinity matrix, using these coupling conditions was 0.75 mg purified avidin/ml of swollen gel. Model stua’ies with puri$ed avidin. 2-Iminobiotin-h-arlinohexyl Sepharose 4B was equilibrated with various buffers with pH ranging from 8.5 to IO.5 Avidin. dissolved in the appropriate equilibrating buffer. was applied to the column and the column isashed with buffer until the absorbance returned to baseline. The hound avidin was eluted with 50 11lM ammonium acetate, pH J. containing 100 mu NaCI. :l,ffinit~~ i.u)tation oj‘u\~iditl. Homogenized egg whites from 24 fresh jumbo eggs were diluted with H20 (2: I. v/v) and the solution \+;I> brought to 70”;’ saturation with anmonium sulfate (enr.yme grade. Schwartz/ Mann) at J’C’. After stirring for 2 h. the mixture wa c,entrifuged (8000~, 20 min). the supernatant brought to 100%) saturation. and the mixture left stirring at 4°C overnight. The solution was centrifuged (SOOOg,
2-IMINOBIOTI~
93
30 min), the pellet dissolved in H,O (40 ml) and dialyzed against Hz0 (3 X 2 liters). The pH of the dialysate was adjusted to 1 I with I N NaOH and NaCl (1 M) added. The crude avidin solution was applied to 2-iminobiotin-6-aminohexyl-Sepharose 4B (40 ml) which had previously been equilibrated with 50 mM sodium carbonate, pH I I, containing I M NaCl. The column was washed with equilibrating buffer (20 ml/h) until the absorbance at 282 nm returned to baseline. Avidin was eluted from the column with 50 mM ammonium acetate, pH 4, containing 0.5 M NaCI. Protein content was measured by absorbance at 282 nm and avidin content by its ability to bind either 4-hydroxyazobenzene-2’-carboxylic acid or [ 14C]biotin ( 16). The appropriate fractions were pooled, dialyzed against HzO, and lyophilized. “‘I-:l\idin. Avidin ( I5 pg) in 40 rn%t borate. pH 8.0 (50 ~1). was added to 100 &i Bolton Hunter reagent and left at 4°C for 60 min. The reaction was terminated by the addition of 200 mM glycine in 40 mM borate. pH 8.2 ( 150 ~1). After 20 min at 4°C. 50 mM ammonium carbonate, pH I I. containing 0.5 M NaCI and 1 mg/ml BSA’ (7 ml) was added. The iodinated avidin was purified as described for avidin. except that all buffers contained BSA ( 1 mg/ml). The column siTe was 4 ml and I .5-ml fractions Mere collected. “‘I-Avidin ~3s stored fro/en
at -20°C. Rllor/anlinr~~a~,itlin.
To ;I solution of avidin (4 mg. 5.9 X IO-’ mol) dissolved in 100 mM sodium bicarbonate, pH 8. (5 ml), at 4°C was added 25 ~1 of rhodamine B isothiocyanate (0.25 mg. 4.7 X 10 ’ 11101) dissolved in ;Y.;Y-dimethylformamide. The reaction was left at 3°C for 15 h and then puritied as described for avidin. The column sixc was 45 ml and 6-ml fractions were collected. Avidin concentration was determined with [ “C’lbiotin and the number of rhoda-
94
HENEY
mine groups introduced was calculated the method of Shechter et al. (4). RESULTS
AND
by
DISCUSSION
Green has published a procedure for the isolation of large amounts of purified avidin ( 18). The method makes use of the high isoelectric point of avidin and involves the absorption of the basic proteins of egg whites onto CM-cellulose at high pH, followed by their elution with a stepwise gradient of ammonium carbonate (0.4 to l.O%,). Rechromatography on CM-cellulose followed by crystallization afforded highly purified avidin. This procedure, although efficient, is time consuming, and also cannot be used for the isolation of biologically active avidin derivatives. Cuutrecasas and Wilchek have developed an affinity isolation procedure for avidin using biocytin (biotin-t-i\i-lysine) coupled to Sepharose 4B (10). The binding of the protein to the affinity matrix is so tenacious that elution can be achieved only by partial denaturation using 6 M guanidine HCI, pH I .5, followed by renaturation after dilution. Avidin, since it is an extremely stable protein, will withstand these conditions. It is likely that certain avidin derivatives. e.g., ferritin-labeled avidin, would lose their desired biological activity by these elution conditions. We have found (12). in agreement with the observations of Green ( I I ), that the free base form of 2-iminobiotin forms a stable complex with avidin, but that the salt form interacts poorly with its binding protein. Our studies indicate that the decrease in affinity observed at neutral and acidic pH values is due to the combined protonation of the cyclic guanidino group of 2-iminobiotin and the ionization of some residue on avidin (12). We have used this pH-dependent alteration in binding to develop an efficient affinity isolation procedure for avidin and its derivatives. Independently, Hofmann and co-worders ( 19) have described the purification of streptavidin. a biotin-binding protein from
AND
ORR
avidinii using the same principle. 2-Iminobiotin is coupled to 6-aminohexyll Sepharose 4B using a water-soluble carbodiimide. Although 2-iminobiotin contains a potentially reactive guanidino group (pk, I Ill 2), this does not complicate the coupling, since the reaction is carried out at pH 4.8 where the group is fully protonated. Purified avidin is bound by the affinity matrix at pH 11 and is eluted as a sharp peak when the pH is lowered to 4 (data not shown). Biotin-treated avidin is not retained by the affinity column, indicating that binding is dependent on the 2-iminobiotin moiety. For efficient binding to immobilized 2-iminobiotin, the pH must be greater than 9.5. In all cases the recovery of avidin was greater than 95%. We have used this aftinity matrix for the isolation of avidin from homogenized egg whites in a single step (data not shown). However, for the purification of large amounts of avidin. we have found it more convenient to carry out an initial ammonium sulfate fractionation. Homogenized egg whites are first brought to 70%’ saturation at 4°C and finally to 100% saturation at which point the majority of the avidin is precipitated. After dialysis the sample is applied to the column at pH I I and the nonspecifically bound proteins are removed by washing the column with the same buffer containing I M NaCI. Avidin is eluted as ;1 sharp peak after application of the low pH buffer (Fig. 1). Greater than 90% of the crude avidin applied to the column is recovered in the specifically eluted fractions and the yield of avidin from 24 eggs is in the range of 15520 mg. We have used the same affinity column for several such avidin preparations with no apparent loss in activity. The avidin obtained by this procedure is pure as judged by its ability to bind 14.4 kg of [‘4C]biotin/mg of protein. Literature values for pure avidin range from 13.8 to IS. I ( I, IO,1 8). SDS polyacrylamide gel electrophoresis (Fig. 2) of the specitically eluted Streptom)ves
AVIDIN
,^
/
i,
iI 1
/ /
PURIFICATION
ON
IMMOBILIZED
I /
FIN;.
3. Purification
bili7ed 2-iminobmtin. for experimental FI(, I. Affinity atIn-6-aminohsxyl luadcd on HI pH plicarion od> for
95
2-IMINOBIOTIN
of pt! details.
purification of avidin on 2-iminobiSepharuse 4B. Crude avidin was I I and specifically eluted by the BP‘1 buffer.
See under
Materials
and
Meth-
fractions reveals a single polypeptide with an apparent Imolecular weight slightly larger than the hemoglobin monomer ( 16,000). Native avidin (68,000) is composed of four identical subunits (1 ). As a further indicrrtion of purity, if the specifically eluted protein is treated with biotin and rechromatographed on the affinity column, no protein is retained 2nd eluted at pH 4 (data not shown ). Although ,lvidin is 3 very stable protein to extremes of pH and temperature and to -
48K
-
32K
-
1-1~; 2. SDS polyclcrylamidr trl’purllicd avidin. The stacking tained 3.5 and IS’/; acrylamide. i/cd hemoglobin subunits (Sigma) ular weight markers. Proteins Cuoma\ic brilliant blue R250.
16K
gel electrophoresis and separating respectively.
( 17) gels conPolymer-
were used as molecwere visualized with
01‘ rhudumine See under
Matcrialh
avidin
on and
immoMethods
details.
proteolytic digestion, it loses biological activity rapidly in the presence of oxidizing agents. e.g., Hz02 and N-bromosuccinimide (I ). In addition, avidin contains only a single, deeply buried, tyrosine residue per subunit, For these reasons. we elected to iodinate avidin using ‘2LI-labeled Bolton Hunter reagent. This reagent acylates primary amino groups, of which 60% in avidin can be blocked without loss of biotin-binding activity (1 ). Avidin is readily iodinatcd by this reagent at pH 8 and retains full biological activity as judged by its ability to bind to and be specifically eluted from the immobilized 2-iminobiotin column (data not shown). Approximately 36% of the initial “‘I was found in the specifically eluted t’ractions and greater than 95’;;’ of the “$1 in these fractions could be precipitated by 105% trichloroacetic acid. We have also used this affinity isolation procedure for the puritication of rhodamineconjugated avidin. Interestingly. the fluorescent avidin derivative does not elute as a sharp peak, 3s observed with the native or iodinated protein, but with 3 long trailing edge (Fig. 3). When the ratio of rhodamine to avidin is calculated along the specitically eluted peak it is found that the ratio increases. This phenomenon allows the aeptlration of rhodamine avidin conjugates with well defined extents of dye incorporation. The pH-dependent interaction of 2-iminobiotin with avidin extends the usefulness of the avidin biotin technology, currently being employed in many areas of molecular
96
HENEY
AND
(7). It will now be possible to selectivelv retrieve a 2-imino-biotin “tagged” polypeptide from a complex mixture of proteins. We have recently demonstrated this potential application by tagging the membrane sialoglycoproteins of the intact human erythrocyte by sequential periodate oxidation/I-iminobiotin hydrazide labeling and retrieving the 2-iminobiotinylated sialoglycoproteins uncontaminated by other membrane or cytosolic proteins ( 12). biology
ACKNOWLEDGEMENT This work was supported in part by Grant GM 27851-01 from the National Institute of Health. REFERENCES I.
Green. 85-l
7. Bayer.
N. M. 13.
(1975)
E.
and
A.,
Biorhrm. 3. Bayer.
FEBS 4. Shechter.
Advan.
ORR
7.
Wilchek,
M.
C‘hm?;.
(197X)
8.
L&t. Y.,
M.. and
Skutelsky.
E. (1976)
J.. Jacobs.
S.. Chnng,
P. (197X)
Pror.
SC?. USA
IL. M..
Davidson.
J Cell
J. F. (1977) Yen.
M. ( 1978)
P. tl..
Hershey.
Rr.c.
,Y~rc,l. .Arfd
P.. and Wilchck, M. (I 968) Biot hen!. Biophys. He\, (‘on~m~or. 33, ‘35-239. 1 I. Green. N. M. (1968) Biochcm J. 101. 774-780. 12. Orr. G. A. ( 1980) J. Bit)/. C‘hertr.. in pres 13. Hofmann. K., and Axelrod. A. E. (1950) J. Biol. C‘hem. 187. 29.. 33. IO. Cuatrecasas.
14.
Hofmann,
(1941) 15.
Porath,
K., Melville.
J. Biol. J.
(McCormick, Vol. 188,
17.
(1974) pp,
D. B.. and
C‘hrnr.
duVlgneaud.
141. ‘07-21
irr
Methods
in
V.
I Ewymologq
D. B.. and Wright. L. D.. eda.), I3 -30. Academic Prws, New York.
Green. N. M. (1970) itr Methods in t~rqrnology (McCormick. D. B.. and Wright. I.. D., eds.), Vol. l8A. pp. 41X 424. Academic Press. New York. Laemmli.
,Yarurr
CJ. K. (1970)
(London/
227. 6X0-
IX.
Green.
N. M..
and
Toms,
E. J. (1970)
Bior,hmt.
J.
118, 67 -70.
Schlcssinger.
Acad.
Ash.
Prw.
685.
Caulrecasas.
;ind
and
(1974)
3541.
5. 363 384.
68, 240-244.
K.-J..
tl..
T. R.. Angerer.
M. D.. and
SC;. 3, N257-259.
E. A.. Wilchek.
M.
73, 783-7xX.
J Suprcrnrvl
F. M.
SC?. C’S,4 71, 3537
Biul.
Sri. US.4 77,
A. E.. ( 1978)
9, 243 -252. H., tend Richards.
Heggeness.
9. Broker.
29,
Twnds
.Slr~cc.f. Hcizmann,
&‘a!. Acad.
16.
Prorrin
Nar. Acad.
5. Axclrod. D. ( 1980) Proc. 4823-4827. 6. Meier, K. E., and Ruoho.
77, 4823-4827.
Nut.
19.
Hofmann. tibeller.
Acad.
K..
Wood.
J. A.. and
S. W.. Finn.
Brinton.
F. M. (1980)
Sc~i. LISA 77, 4666-4668.
C. C., Pntt,.
Mon:Var.
Purification
of Avidin
and
Its Derivatives
2-lminobiotin-6-aminohexyl-Sepharose GAYLE
HENEY
Received
and
The pH-dependent avidin has been
interaction used in the
its fluorescent and iodinated of 2-iminobiotin-6-aminohexylSepharose ically eluted from the column conditions, i.e., 6 M guanidineeHC1. biotin
AND
0003-2697/8
A.
16.
derivatives. at pH
Avidin and 48 at pH 4. This affinity pH 1.5. required
ORR’
I980
between the cyclic guanidino design of an efficient affinity
analog isolation
its derivatives values between
of biotin. system
2.iminobiotin. for avidin
and
are retained by a column 9 and I I and are spectf-
isolation procedure to dissociate avidin
overcomes the harsh from an immobilkd
column.
The avidin biotin complex is characterized by an extremely low dissociation constant (Kn) of approximately lo-15 M (1). The tightness of this interaction has formed the basis for the useof the complex in several cytochemical techniques in membrane and molecular biology (2). For example, avidin biotin complex formation has been used successfully for the visualization and quantitication of receptors for lectin (3) polypeptide hormones (4). and other molecules (5,6) on the cell surface. Fluorescent and electrondenseavidin derivatives have been employed to observe biological membranes labeled with biotin (7) and for the ultrastructural localization of biotinylated intracellular components (8). Davidson’s group has described methods for electron microscopic gene mapping using specfic RNA biotin cytochrome c and avidin -ferritin complexes to visualize RNA -DNA hybridization (9). In many of these studies it would be desirable if an affinity isolation system were available for the isolation of biologically active avidin derivatives uncontaminated by damaged or unconjugated avidin molecules. Although the essentially covalent character of the nvidin biotin complex makes it an ideal choice for many of the above mentioned studies, it severely restricts its use for ’ To whom
46
GEORGE
December
on
all
correspondence
should
I /090092-05$02.00/0
Copyrnght c INI by Acddemx Prcsr. Inc. All right, of repruducuon ,n any form reserved
be addressed.
the affinity isolation of (a) avidin and its derivatives and (b) biotinylated components. Cuatrecasas and Wilchek have reported the isolation of avidin on a biocytin (biotin-t-IVlysyl)Sepharose 4B column ( IO). The conditions required for the elution of the specifically bound protein were a combination of low pH (1.5) and 6 M guanidine HCI: either alone did not effect elution. Although avidin is not inactivated by these harsh elution conditions, it is uncertain whether certain avidin derivatives. e.g., ferritin-conjugated avidin, would retain their desired biological activity. We have overcome this problem by replacing biotin with its cyclic guanidino analog. 2-iminobiotin. At high pH (>9) 2-iminobiotin exists primarily in its free base form and retains the tight specific binding to avidin characteristic of biotin ( I I. 17). At low pH, where it exists primarily in the salt form. it interacts weakly with avidin. This paper describes the synthesis of 7-iminobiotin-6-aminohexyl--Sepharose 4B and its use for the affinity isolation of avidin and of its biologically active fluorescent and iodinated derivatives. MATERIALS
AND METHODS
d-Biotin and avidin were obtained from Sigma Chemical Company, St. Louis, Mis-
AVIDIN
PURIFICATION
ON
IMMOBILIZED
souri. ci-[~arho,!l,/-“C]Biotin (56 mCi/ mmol) and “‘I-labeled Bolton Hunter reagent (1500 Ci/mmol) were from Amersham. Rhodamine B isothiocyanate was purchased fron! ICN. The cyclic guanidino analog of biotin, _‘-iminobiotin. was synthesized from the diamino carboxylic acid derivative of biotin, S-( 3,4-diamino-thiophan2-yl) pentanoic acid by reaction with cyanogen bromide (13,14).
,7-lminobiotin-6-aminohexyl-Sepharose di3. 2-lminohiotin (400 mg. 1.23 mmol) was added to 100 ml of 6-aminohexyl Sepharose 4B (40 ml of packed resin in H,O). prepared by the method of Porath ( 15). and the pH was adjusted to 4.8 with HBr ( I%, v/v). 1-Cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (4.24 g, 10 mmol) was added portionwise over a period of IO min. The pH was kept at 4.8 throughout the reaction by the addition of 1% H Br and was constant after 3 to 5 h. The resin was washed with I M NaCl (2 liters), H,O (2 liters) and packed into a column. The binding capacity of the affinity matrix, using these coupling conditions was 0.75 mg purified avidin/ml of swollen gel. Model stua’ies with puri$ed avidin. 2-Iminobiotin-h-arlinohexyl Sepharose 4B was equilibrated with various buffers with pH ranging from 8.5 to IO.5 Avidin. dissolved in the appropriate equilibrating buffer. was applied to the column and the column isashed with buffer until the absorbance returned to baseline. The hound avidin was eluted with 50 11lM ammonium acetate, pH J. containing 100 mu NaCI. :l,ffinit~~ i.u)tation oj‘u\~iditl. Homogenized egg whites from 24 fresh jumbo eggs were diluted with H20 (2: I. v/v) and the solution \+;I> brought to 70”;’ saturation with anmonium sulfate (enr.yme grade. Schwartz/ Mann) at J’C’. After stirring for 2 h. the mixture wa c,entrifuged (8000~, 20 min). the supernatant brought to 100%) saturation. and the mixture left stirring at 4°C overnight. The solution was centrifuged (SOOOg,
2-IMINOBIOTI~
93
30 min), the pellet dissolved in H,O (40 ml) and dialyzed against Hz0 (3 X 2 liters). The pH of the dialysate was adjusted to 1 I with I N NaOH and NaCl (1 M) added. The crude avidin solution was applied to 2-iminobiotin-6-aminohexyl-Sepharose 4B (40 ml) which had previously been equilibrated with 50 mM sodium carbonate, pH I I, containing I M NaCl. The column was washed with equilibrating buffer (20 ml/h) until the absorbance at 282 nm returned to baseline. Avidin was eluted from the column with 50 mM ammonium acetate, pH 4, containing 0.5 M NaCI. Protein content was measured by absorbance at 282 nm and avidin content by its ability to bind either 4-hydroxyazobenzene-2’-carboxylic acid or [ 14C]biotin ( 16). The appropriate fractions were pooled, dialyzed against HzO, and lyophilized. “‘I-:l\idin. Avidin ( I5 pg) in 40 rn%t borate. pH 8.0 (50 ~1). was added to 100 &i Bolton Hunter reagent and left at 4°C for 60 min. The reaction was terminated by the addition of 200 mM glycine in 40 mM borate. pH 8.2 ( 150 ~1). After 20 min at 4°C. 50 mM ammonium carbonate, pH I I. containing 0.5 M NaCI and 1 mg/ml BSA’ (7 ml) was added. The iodinated avidin was purified as described for avidin. except that all buffers contained BSA ( 1 mg/ml). The column siTe was 4 ml and I .5-ml fractions Mere collected. “‘I-Avidin ~3s stored fro/en
at -20°C. Rllor/anlinr~~a~,itlin.
To ;I solution of avidin (4 mg. 5.9 X IO-’ mol) dissolved in 100 mM sodium bicarbonate, pH 8. (5 ml), at 4°C was added 25 ~1 of rhodamine B isothiocyanate (0.25 mg. 4.7 X 10 ’ 11101) dissolved in ;Y.;Y-dimethylformamide. The reaction was left at 3°C for 15 h and then puritied as described for avidin. The column sixc was 45 ml and 6-ml fractions were collected. Avidin concentration was determined with [ “C’lbiotin and the number of rhoda-
94
HENEY
mine groups introduced was calculated the method of Shechter et al. (4). RESULTS
AND
by
DISCUSSION
Green has published a procedure for the isolation of large amounts of purified avidin ( 18). The method makes use of the high isoelectric point of avidin and involves the absorption of the basic proteins of egg whites onto CM-cellulose at high pH, followed by their elution with a stepwise gradient of ammonium carbonate (0.4 to l.O%,). Rechromatography on CM-cellulose followed by crystallization afforded highly purified avidin. This procedure, although efficient, is time consuming, and also cannot be used for the isolation of biologically active avidin derivatives. Cuutrecasas and Wilchek have developed an affinity isolation procedure for avidin using biocytin (biotin-t-i\i-lysine) coupled to Sepharose 4B (10). The binding of the protein to the affinity matrix is so tenacious that elution can be achieved only by partial denaturation using 6 M guanidine HCI, pH I .5, followed by renaturation after dilution. Avidin, since it is an extremely stable protein, will withstand these conditions. It is likely that certain avidin derivatives. e.g., ferritin-labeled avidin, would lose their desired biological activity by these elution conditions. We have found (12). in agreement with the observations of Green ( I I ), that the free base form of 2-iminobiotin forms a stable complex with avidin, but that the salt form interacts poorly with its binding protein. Our studies indicate that the decrease in affinity observed at neutral and acidic pH values is due to the combined protonation of the cyclic guanidino group of 2-iminobiotin and the ionization of some residue on avidin (12). We have used this pH-dependent alteration in binding to develop an efficient affinity isolation procedure for avidin and its derivatives. Independently, Hofmann and co-worders ( 19) have described the purification of streptavidin. a biotin-binding protein from
AND
ORR
avidinii using the same principle. 2-Iminobiotin is coupled to 6-aminohexyll Sepharose 4B using a water-soluble carbodiimide. Although 2-iminobiotin contains a potentially reactive guanidino group (pk, I Ill 2), this does not complicate the coupling, since the reaction is carried out at pH 4.8 where the group is fully protonated. Purified avidin is bound by the affinity matrix at pH 11 and is eluted as a sharp peak when the pH is lowered to 4 (data not shown). Biotin-treated avidin is not retained by the affinity column, indicating that binding is dependent on the 2-iminobiotin moiety. For efficient binding to immobilized 2-iminobiotin, the pH must be greater than 9.5. In all cases the recovery of avidin was greater than 95%. We have used this aftinity matrix for the isolation of avidin from homogenized egg whites in a single step (data not shown). However, for the purification of large amounts of avidin. we have found it more convenient to carry out an initial ammonium sulfate fractionation. Homogenized egg whites are first brought to 70%’ saturation at 4°C and finally to 100% saturation at which point the majority of the avidin is precipitated. After dialysis the sample is applied to the column at pH I I and the nonspecifically bound proteins are removed by washing the column with the same buffer containing I M NaCI. Avidin is eluted as ;1 sharp peak after application of the low pH buffer (Fig. 1). Greater than 90% of the crude avidin applied to the column is recovered in the specifically eluted fractions and the yield of avidin from 24 eggs is in the range of 15520 mg. We have used the same affinity column for several such avidin preparations with no apparent loss in activity. The avidin obtained by this procedure is pure as judged by its ability to bind 14.4 kg of [‘4C]biotin/mg of protein. Literature values for pure avidin range from 13.8 to IS. I ( I, IO,1 8). SDS polyacrylamide gel electrophoresis (Fig. 2) of the specitically eluted Streptom)ves
AVIDIN
,^
/
i,
iI 1
/ /
PURIFICATION
ON
IMMOBILIZED
I /
FIN;.
3. Purification
bili7ed 2-iminobmtin. for experimental FI(, I. Affinity atIn-6-aminohsxyl luadcd on HI pH plicarion od> for
95
2-IMINOBIOTIN
of pt! details.
purification of avidin on 2-iminobiSepharuse 4B. Crude avidin was I I and specifically eluted by the BP‘1 buffer.
See under
Materials
and
Meth-
fractions reveals a single polypeptide with an apparent Imolecular weight slightly larger than the hemoglobin monomer ( 16,000). Native avidin (68,000) is composed of four identical subunits (1 ). As a further indicrrtion of purity, if the specifically eluted protein is treated with biotin and rechromatographed on the affinity column, no protein is retained 2nd eluted at pH 4 (data not shown ). Although ,lvidin is 3 very stable protein to extremes of pH and temperature and to -
48K
-
32K
-
1-1~; 2. SDS polyclcrylamidr trl’purllicd avidin. The stacking tained 3.5 and IS’/; acrylamide. i/cd hemoglobin subunits (Sigma) ular weight markers. Proteins Cuoma\ic brilliant blue R250.
16K
gel electrophoresis and separating respectively.
( 17) gels conPolymer-
were used as molecwere visualized with
01‘ rhudumine See under
Matcrialh
avidin
on and
immoMethods
details.
proteolytic digestion, it loses biological activity rapidly in the presence of oxidizing agents. e.g., Hz02 and N-bromosuccinimide (I ). In addition, avidin contains only a single, deeply buried, tyrosine residue per subunit, For these reasons. we elected to iodinate avidin using ‘2LI-labeled Bolton Hunter reagent. This reagent acylates primary amino groups, of which 60% in avidin can be blocked without loss of biotin-binding activity (1 ). Avidin is readily iodinatcd by this reagent at pH 8 and retains full biological activity as judged by its ability to bind to and be specifically eluted from the immobilized 2-iminobiotin column (data not shown). Approximately 36% of the initial “‘I was found in the specifically eluted t’ractions and greater than 95’;;’ of the “$1 in these fractions could be precipitated by 105% trichloroacetic acid. We have also used this affinity isolation procedure for the puritication of rhodamineconjugated avidin. Interestingly. the fluorescent avidin derivative does not elute as a sharp peak, 3s observed with the native or iodinated protein, but with 3 long trailing edge (Fig. 3). When the ratio of rhodamine to avidin is calculated along the specitically eluted peak it is found that the ratio increases. This phenomenon allows the aeptlration of rhodamine avidin conjugates with well defined extents of dye incorporation. The pH-dependent interaction of 2-iminobiotin with avidin extends the usefulness of the avidin biotin technology, currently being employed in many areas of molecular
96
HENEY
AND
(7). It will now be possible to selectivelv retrieve a 2-imino-biotin “tagged” polypeptide from a complex mixture of proteins. We have recently demonstrated this potential application by tagging the membrane sialoglycoproteins of the intact human erythrocyte by sequential periodate oxidation/I-iminobiotin hydrazide labeling and retrieving the 2-iminobiotinylated sialoglycoproteins uncontaminated by other membrane or cytosolic proteins ( 12). biology
ACKNOWLEDGEMENT This work was supported in part by Grant GM 27851-01 from the National Institute of Health. REFERENCES I.
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