BIOTIN AND DERIVATIVES
 Spectrophotometric D e t e r m i n a t i o n of Avidin and Biotin
By N. M. GREEN Since avidin combines stoichiometrically with biotin, it is possible to use any physicochemical difference between avidin and the avidin-biotin complex as the basis of an assay method for either component. Two convenient techniques have been described. The first is based on the red shift of the absorption spectrum of the tryptophan residues of avidin, which accompanies combination with biotin, and requires measurement of the change in optical density at 233 nm. 1 The second is based on the use of the dye 4-hydroxyazobenzene-2'-carboxylic acid (HABA), which binds only to avidin and can therefore be used as an indicator for unoccupied binding sites.: The method based on spectral shift is technically more demanding and has no advantages over the dye-binding method for routine estimations, but it is useful for the study of biotin analogs. Both methods are of lower sensitivity than the bioassays, but they are more precise, more convenient, and can be used over a wide range of pH and salt concentration. Methods for determination of avidin based on the use of radioactive biotin are described elsewhere (this volume ). The unit of biotin binding activity is the amount of protein which binds 1 ug of biotin. Reagents
Avidin (Worthington, 12-13 units/mg; Nutritional Biochemicals, 2-3 units/rag) or biotin 4-Hydroxyazobenzene-2'-carboxylic acid (HABA) (Eastman Kodak, Koch-Light). The dye should be recrystallized from aqueous methanol since impure preparations have given anomalous results at high salt concentration. One equivalent of base is required to dissolve it in water. Buffer, e.g., 0.1 M sodium phosphate, pH 7. Any other convenient buffer from pH 4-8 of any ionic strength can be used. The binding of the dye HABA by avidin is accompanied by spectral changes summarized in Table I. More detailed spectral characteristics of the dye may be found elsewhere2 HABA is not bound by the avidin-biotin N. M. Green, Biochem. J. 89, 585 (1963). 2 N. M. Green, Biochem. J. 94, 23C (1965). 3 j. Baxter, Arch. Biochem. Biophys. 108, 375 (1964).
AVIDIN AND BIOTIN
complex, and since the dissociation constant of the latter is so low (10 -15 M) the dye is stoichiometrically displaced by biotin. This can be made the basis of b o t h colorimetric and titrimetric assays. TABLE I EXTINCTION COEFFICIENTS OF 4-HYDROXYAZOBENZENE-2t-CARBOXYLIC ACID AND ITS COMPLEXI~]S WITH AVIDIN AND STHEPTAVIDINa
Avidin HABA Avidin-HABA complex Streptavidin Streptavidin-HABA complex
282 350 500 280 500
25,000 2,800 -(57,000) b --
0 20,500 2,000 ---
0 480 35,500 0 35,000
a All extinction coefficients are expressed per mole of biotin bound. b This figure is corrected for slight opalescence of streptavidin solutions. Colorimetric Assay for Avidin 1. To 2.0 ml of avidin solution (0.1-1 mg protein in 0.1 M phosphate or acetate buffer at any p H between 4 and 8) in a l-era cuvette, add H A B A (10 raM, 50 ul). 2. Measure Asoo. 3. Add biotin (2 mM, 50 ul). 4. Measure Asoo to calculate [binding sites] = AAs00 34 m M and avidin ( m g / m l original solution) = ~2"05 × ~16"2 AA~oo = 0.49 AAsoo Colorimetric Procedure for Biotin 1. Pipette 2.0 ml of a solution of a v i d i n - H A B A complex (0.2~).4 mg a v i d i n / m l 0.25 m M H A B A in 0.1 M phosphate buffer or acetate buffer, p H 4-8) into a l-era cuvette. 2. Measure Asoo (Aj). 3. Add a known volume of biotin (v, ml) and again measure As0o (A~) to calculate [biotin] =
A1 - A2(v q-- 2)/2 m M 34
BIOTIN AND DERIVATIVES
P r o v i d e d t h a t v < 0.2 ml, t h e f a c t o r (v + 2)/2 has a negligible effect on AAs00 a n d m a y be ignored. I f v > 1.0 ml, c o r r e c t i o n is necessary for d i l u t i o n of t h e d y e as i n d i c a t e d in T a b l e I I . W h e n v is negligible, m i c r o g r a m s of b i o t i n p e r milliliter = 7.2 AAs00, or 1 ~g b i o t i n in 2 ml gives AAs00 of 0.069. I t is possible to use a solution of p u r e b i o t i n as a s t a n d a r d in place of t h e factors given. V a r i a t i o n s of C o n d i t i o n s 1. Use of m i c r o c u v e t t e s increases t h e s e n s i t i v i t y . 2. T h e m e t h o d can be used over a wide r a n g e of p H a n d s a l t concent r a t i o n . Below p H 4 t h e d y e begins to p r e c i p i t a t e , since its c a r b o x y l g r o u p is p r o t o n a t e d ( p K , ~ 4), a l t h o u g h b i n d i n g ~o a v i d i n is n o t m u c h affected u n t i l t h e p H is below 2. A b o v e p H 8 the h y d r o x y l g r o u p of H A B A ionizes w i t h a p K of 8.5, a n d t h e ionized form does n o t b i n d to a v i d i n w i t h a c c o m p a n y i n g spectral change. T h e a p p a r e n t dissociation c o n s t a n t of t h e d y e t h e r e f o r e increases r a p i d l y a b o v e p H 7 ( T a b l e I I ) . B e t w e e n p H 4 a n d 8, t h e m e t h o d can be used as described, since t h e e x t i n c t i o n coefficient is i n d e p e n d e n t of p H , a n d t h e d i s s o c i a t i o n c o n s t a n t varies o n l y s l i g h t l y ( T a b l e I I ) . T h e m e t h o d has been used m o s t l y w i t h a c e t a t e or p h o s p h a t e buffers, b u t i t a p p e a r s insensitive to t h e n a t u r e of t h e buffer or t h e c o n c e n t r a t i o n of salt. TABLE II EFFECT OF pH ON EXTINCTION COEFFICIENTS AND DISSOCIATION CONSTANTS OF THE AVIDIN DYI~ COMPLEX (AI)) a
~,,~ × 10-3 b
K M X 106
4 5 6 7 8 9 7
34 33 34 34 33 23 24
6 13 6 7 12 65 100
a The results for avidin at pH 7 are taken from N. M. Green, Biochem. J. 94, 23C (1965). The remainder are from unpublished experimenls. b A,~0 is an apparent A, calculated from the change in optical density following addition of excess biotin, when the concentration of dye is 0.25 mM. It is equal to A A ~ / mole biotin bound per liter. Under these conditions, the binding sites are not quite saturated: [ADJ/[A total] = D/(D -k K). The true *~o (Table I) = [A,app(D q- K)/D -k 480], where 480 is the extinction coefficient of the free dye. This equation can be used to calculate A*apoat other dye concentrations. It is necessary to use a 15-fold greater dye concentration to achieve comparable saturation of streptavidin.
AVIDIN AND BIOTIN
For example, 0.5 M ammonium sulfate has no effect on K or ~ at pH 7.0, and even 3.5 M ammonium sulfate produces very small efl'ects. 3. Lower concentrations of dye may be used provided that a correction is made for the smaller fraction of sites occupied by dye (Table II). It is inconvenient to use much higher dye concentrations because of the appreciable absorbance of the unbound dye (Table I). Titration Method The solutions used are the same as those employed above, but the biotin is added in small (1-10 ul) aliquots from a Hamilton microsyringe until no further change in As00 is observed. The end point, determined by plotting As00 against biotin added, is sharp, and the equivalence point between given solutions of avidin and biotin can be estimated within a few percent, so that the concentration of one may be determined if that of the other is known. The method is slightly more laborious than the colorimetric method, but it does not require any knowledge of the extinction coefficient or dissociation constant of the avidin-dye complex and can therefore be used over an even wider range of conditions without any corrections. Specificity The only other proteins that have been shown to give color changes with HABA are streptavidin2 and serum albumin. 3 The binding to streptavidin is somewhat weaker (Table II), so that more dye is required to achieve a comparable saturation of the binding sites. 4 If this is taken into account it may be used in place of avidin for the estimation of biotin. Since the dye is not displaced from serum albumin by low concentrations of biotin it will not interfere with the assay, unless it is present in such large amounts that it binds a large fraction of the HABA.
Biotin Analogs Combined forms of biotin, such as biocytin and various biotinyl enzymes (see below), are bound by avidin and titrate as biotin in this system. Biotin analogs will also displace H A B A if the dissociation constants of their complexes are < 10-6 M ; however, they will not give sharp end points unless K < I0-g M. Curvature in the neighborhood of the end point serves to distinguish weakly bound analogs from biotin, biotin sulfone, dethiobiotin, and bioeytin. Information obtained with bifunctional biotin compounds 5 and with biotin linked to an insoluble matrix 6 suggests that biotinyl proteins will 4 N. M. Green, unpublished experiments. 6 N. M. ~reen, Biochem. J. 104, 64P (1967). 6 p. Cuatrecasas and M. Wilchek, Biochem. Biophys, Res. Commun. 33, 235 (1968).
BIOTIN AND DERIVATIVES
react with avidin only when the biotin is separated from the surface of the macromolecule by at least five methylene groups. It is therefore possible that some forms of bound biotin will not be extimated by these methods. For example, the biotinyl groups in the polymeric form of acetyl coenzyme A carboxylase (activated by isocitrate) is not available to avidin. 7 Estimation of Biotin Content of Biotinyl Enzymes 4,8 When concentrated solutions of some biotinyl enzymes are added to avidin-HABA complex, the dye is displaced, but more slowly th~n by free biotin. When more than about 30% of the dye has been displaced, the reaction becomes even slower ~md does not go to completion. It appears that owing to the multi-subunit nature of both avidin and of the enzymes, it is sterically impossible to achieve a stoichiometric reaction. The magnitude of the anomaly varies with the enzyme and is, for example, much greater with pyruw~te carboxylase than with transcarboxylase, which initially reacts fairly rapidly and completely. It is possible to eliminate these problems by digestion of the enzyme with pronase for 24 hours at 37 ° and assaying the digest by the colorimetric method described above, using concentrated solutions of the enzyme digest (2-5%) in order to obtain adequate optical density change. The presence of peptide digestion products does not affect the assay. Spectral Shift Method for Assay of Biotin and Its Analogs ~ The method requires avidin of high specific activity ( > 10 units/rag). The biotin solution should have low absorption in the UV down to 230 nm. Measurements of difference spectra are best made with a recording spectrophotometer. Some of the technical problems that may be encountered are considered in detail elsewhere? 1. Two cuvettes are prepared with identical volumes (e.g., 2.0 ml) of a solution of avidin of A280 < 0.4 (A23~ < 1.6). The difference spectrum (in this case a flat baseline) between 230 and 330 nm is determined with a recording spectrophotometer. 2. The w~velength is set to 233 nm (or 43,000 cm -~) and a 2-10 ~1 sampie of a solution of biotin (or analog) is added to one cuvette from a Hamilton microsyringe. An identical volume of buffer is added to the reference cuvette. 3. After adequate mixing (e.g., by a small magnetic stirrer in the cuvette), the A233 is recorded. 7 E. Ryder, C. Gregolin, C. H. Chang, and M. l). Lane, Proc. Natl. Acad. Sci. U.S. 57, 1455 (1967). 8 M. C. Scrutton and A. S. Mildvan, Biochemistry 7, 1490 (1968). ' T. T. Herskovits, Vol. XI, p. 771.
AVIDIN AND BIOTIN
T A B L E III BINDING OF BIOTIN AND ANALOGS TO AVIDIN AT pH 7 °,b
Compound Biotin Biotin sulfone N-Biotinyl-~-aminohexanoate Biotin anilide Biotin m e t h y l ester 2'-Thiobiotin (pH 9.0) D-Dethiobiotin DL-Dethiobiotin m e t h y l ester 2'-Iminobiotin (free base) N-3'-Methoxycarbonyl biotin m e t h y l ester N - l ' - M e t h o x y c a r b o x y l biotin m e t h y l ester Diamine from biotin (free base) [~-(3,4-diaminothiophan-2-yl)pentanoic acid] DL-3-n-Hexyl imidazolid-2-one DL-3-n-Butyl imidazolid-2-one Di-3-n-Propyl imidazolid-2-one DL-3-Ethyl imidazolid-2-one DL-3-Methyl imidazolid-2-one Imidazolid-2-one (ethylene urea) Urea DL-Lipoate n-Decanoate n-Hexanoate n-Hexyl alcohol
Dissociation c o n s t a n t of complex (M)
0.94 d 0.84 d 0.76 ~ 0.68 ~ 0.74 1.091 0.47 0.39 0.76 0.45 0,52
10 -1~ • < 10-~a J.~ < 10-~3/.0 < 10-13 l.o < 1 0 -~S f.g 5 )< 10-130.h 5 × 10 - ~ J'.0 1 >( 10- u / . g 3 . 5 × 10- u h 10-8 to 10~ 4 >( 10-~ J
0.73 0.41 0.40 0.24 0.22 0.23 0.26 0.33 0.39 0.11i 0.10i 0.09 k
1.2 1 1 5 8 3 5 4 7 1 3 4
X X × X X × X X X X X X
10-~ h 10-7 / 10-6 I 10 -6 ! 10-e / 10-6 / 10-4 * 10-2 a 10-7 d 10-~ ~ 10-4 a 10-a /
Information on the competition between biotin and a n u m b e r of other analogs is given by L. D. Wright, H. R. Skeggs, and E. L. Cresson, Proc. Soc. Exptl. Biol. Med. 64, 150 (1947). b T h e difference extinction coefficients and dissociation constants do not depend strongly on pH between 4 and 10 unless the analog undergoes changes in ionization. M o s t complexes have Xm,x = 233.6 n m (43,000 cm-l). T h e values of AA~,x/A~8, were measured with a Unicam SP,700 recording spectrophotometer. Values some 10-15% higher are found with Cary spectrophotometers on account of their lower stray light level. A+ per mole analog bound = AAm~x/A2s.~ >( 25,000. N. M. Green, Biochem. J. 89, 599 (1963). + N. M. Green, Biochem. J. 89, 585 (1963). / N. M. Green, unpublished experiments. Approximate estimate from the rate of displacement of the analog by biotin. Procedure described for thiobiotin in footnote h. N. M. Green, Biochem. J. 101,774 (1966). i X ~ 238 nm. i k ~ 229 nm. k h~,,., 231 nnl.
BIOTIN AND DERIVATIVES
4. The additions are repeated until no further change occurs. 5. T h e difference spectrum is recorded from 230 to 330 nm, as a check for anonmlous spectral effects. When a particular system is thoroughly characterized, the recording of the difference spectrum may be omitted. 6. A_~33is plotted against the amount of biotin or of analog added and the equivalence point is read from the graph. Sharp end points are obtained provided that K (the dissociation constant of the complex) is < l0 -7 M.
Notes 1. The total A233 at the end point should be <2.0 to avoid any errors from stray light. For this reason, avidin of high purity is required ( > 70%). 2. Volumes must be precisely controlled. 3. If the biotin analog contributes appreciably to A233, this must be corrected either arithmetically or by use of tandem cells containing analog alone, at the same concentration as in the sample cuvette2 4. The value of Ae233,which is most readily expressed as AA233/(A282 due to avidin), is characteristic of a particular analog, as is the profile of the difference spectrum in the 280 nm region. Table III gives values of AA2a~/ A28~ and of the dissociation constants of a number of analogs to give some idea of the compounds that may affect assays involving avidin. The main drawbacks of the method are the exacting technique required for good results and its sensitivity to interference by substances absorbing at 230 nm. Its precision can be nearly as good as that of the HABA methods, and it has advantages in that it can provide information about the dissociation constants of the complexes of avidin with biotin analogs and can serve as a method of characterizing such analogs.
 Assay of Avidin By RU-DONGWEI Definition of Unit and Specific Activity. A unit of avidin is defined as the amount that binds or inactivates 1 tLg of ( + ) - b i o t i n ? Specific activity is expressed as units of avidin per milligram of protein. On this basis, the ' R. E. Eakin, E. E. Snell, and R. J. Williams, J. Biol. Chem. 140, 535 (1941).