Antigen-Antibody Recognition by Fourier Transform IR Spectroscopy/ Attenuated Total Reflection Studies: Biotin-Avidin Complex as an Example R. BARBUCCI,’ A. MACNANI,’ C. RONCOLINI,’ and S. SILVESTRI*
’Dipartimentodi Chimica, Piano dei Mantellini, 44 53100 Siena, and ’SCLAVO S.p.A., Via Fiorentina, 1 53100 Siena, Italy
SYNOPSIS
Biotin-avidin recognition is studied by Fourier transform ir spectroscopy /attenuated total reflection (FTIR/ ATR) under physiological conditions. The ureido portion of biotin is confirmed to be involved in the interaction with avidin, as previously found, but when the biotin-avidin complex forms, an electrostatic interaction occurs between the carboxylate group of the biotin molecule and the protonated aminic end group of the avidin amino acid side chains. Comparison of the biotin-avidin system with the biotin-1,4-diaminobutane and biotin-tryptophan systems confirms these findings.
0
INTRODUCTION
II
It is known that avidin from the whites of hen eggs is a tetramer composed of 15700 M , monomers; each subunit of avidin binds one molecule of D-biotin with a n affinity (pKd 15) among the highest of all associations between naturally occurring ligands and biological macromolecules.’ Binding studies of biotin analogues have shown that the main interaction of the biotin molecule with avidin involves only the ureido portion of the ligand.’.3 In addition, the property of D-biotin to protect tryptophan residues from oxidizing agents suggests the proximity of indole side chains to the binding pocket for biotin.’ In the present study we investigated the biotinavidin system by fourier transform attenuated total reflection ir spectroscopy with the aim of -verifying the “performance” of this spectroscopic technique in analyzing interacting biological systems, and -identifying the chemical groups involved in the interactions of this antigen-antibody recognition. Riopolymer?. Vol. 31, 827-834 (1991) (c, 1991 John Wiley & Sons, Inc.
CCC 0006-3525/91/070827-08504.00
HOOC(CH,),-HC,
,CH2 S
Scheme 1. Structure of biotin.
EXPERIMENTAL Materials
+
-Lyophilized crystalline D ( ) biotin was used a s received from Merck. -Avidin was used a s received from Societii P r o dotti Antibiotici. -L-Tryptophan amino acid was used as received from Sigma Chemical Company and 1,4-diaminobutane as received from Aldrich Chemical Company. Each protein solution was prepared by dissolving the powder in a phosphate buffer solution, and the p H was adjusted by adding small amounts of aqueous HC1 or NaOH solutions. The solution concentrations were 1.5 10-3M for biotin, 1.5 10 3 M for av-
-
-
827
828
BARBUCCI ET AL.
idin, 1.0.10-2M for tryptophan, and 1.0. for 174-diaminobutane. The biotin-avidin complex solution was obtained by mixing the two components in the ratio 4 : 1 ( M / M) biotin/avidin. The biotin-tryptophan and biotin-1,4-diaminobutane systems were obtained by mixing biotin with an excess of tryptophan or 1,4-diaminobutane. Instrumentation
The spectra were obtained with a Perkin-Elmer Fourier transform ir (FTIR) spectrometer M 1800 operating between 3000 and 900 cm-’ . An MCT detector was used and the apparatus purged with dry nitrogen. Typically 300 scans at a resolution of 2.0 cm-’ were averaged and the spectra stored on floppy disks. The frequency scale was internally calibrated with a reference He-Ne laser to an accuracy of 0.01 cm-’ . A Barnes microcircle cell for liquid with a germanium crystal was used to record the spectra in solution. Spectral Processing
All the spectra in solution were taken in the single beam mode. The sample spectrum was obtained subtracting the saline solution spectrum from the sample solution spectrum. The scale factor for saline solution subtraction was chosen so that the spectral region between 2000 and 1700 cm-’ was flat. Biotin and Biotin-Tryptophan/ 7,4-Diaminobutane Systems. The difference spectra for these species in
solution were obtained directly by subtracting the spectrum of the saline solution from that of the sample solution. Avidin and Biotin-Avidin Complex. When a protein
solution is introduced into the ATR cell, there is immediate protein adsorption on the surface of the internal reflection element, so that the spectrum of the sample solution consists of two contributions: that of the adsorbed protein and that of the “bulk” protein. The term “bulk” protein refers to the protein in solution but only of layer having a thickness equal to the penetration depth of the ir beam. In order to obtain the spectrum of the bulk protein, the spectrum obtained has to be corrected for the contribution of the adsorbed protein. The spectra of the protein solutions ( avidin or biotin-avidin complex) were therefore recorded after different periods of exposure (adsorption experiment). By sub-
tracting the spectrum of the saline solution from those of the protein solution, the spectra of the (bulk adsorbed) protein at different times were obtained. When a constant spectrum was obtained, the protein solution was replaced with fresh saline solution and the spectra were collected until a constant spectrum was again achieved (desorption experiment). This spectrum represented the adsorbed protein. Subtraction of this spectrum from the one collected at the end of the adsorption experiment gave the spectrum of the bulk protein.
+
+
Spectrum “Sum” (Biotin Avidin). The addition spectrum was obtained by collecting the spectra of biotin and avidin in solution at the same molar concentrations as in the complex, and then summing the two. Spectra of Biotin Interacting with Avidin, Tryptophan, and l,4-Diaminobutane. The difference
spectra of the interacting biotin were obtained by subtracting the spectra of the native avidin, tryptophan, or 1,4-diaminobutane from those of the corresponding systems involving biotin. The criterion adopted was to minimize a pure band of the component to be subtracted. Spectral Enhancement. In order to improve the ob-
servability of the overlapping bands, mathematical resolution enhancement was performed by a spectral deconvolution process that is like the Fourier selfdeconvolution, except that the mathematical operations are performed in the spectral domain rather in the Fourier domain. The spectral deconvolution process moves intensity from the outer wings of a band into the center of the band, thus reducing its effective half width and improving its o b s e ~ a b i l i t y . ~ The quality of the deconvolution procedure is controlled by two variables, namely half-band width of the Lorentzian line used for deconvolution and the resolution enhancement achieved. 536
RESULTS AND DISCUSSION Avidin
The spectra of the protein in normal saline ( p H 7.40) collected after different periods of adsorption are reported in Figure 1. The ratio of band height at 1540 and 1528 cm-’ (correlated to the amount of the protein) increases from A (0.93) to B (0.97), to C (1.02), whereas it remains unchanged from C to D ( 1.02). Thus spectra C and D can be regarded as relative to the protein
ANTIGEN-ANTIBODY RECOGNITION
829
The higher intensity of the bands of the @sheet conformation with respect t o the a-helix confirms that in physiological solution a higher percentage of the amino acids in avidin participate in P-sheet conformation than in a-helix conformation. This agrees with the data reported in the literature ( 10% a-helix, 55% &sheet) .399
Biotin
t
--Wd o i l !
i
i
I
I
1653
I
cm'
I
I
I
I
I'
0 0
i-153
Figure 1. Difference spectra of avidin (bulk + adsorbed protein ) collected after different periods of adsorption: ( A ) 1 min, ( B ) 1 h, ( C ) 3 h, and ( D ) 24 h.
in the steady state. These protein spectra consist of the contribution of both the adsorbed and bulk protein and the changes observed within the first 3 h of exposure are presumably related to diffusion and adsorption p r o c e ~ s e s .When ~ the steady state is reached the spectrum of the adsorbed protein may be collected (see experimental section). By subtracting this spectrum from the spectrum C ( o r D ) , the spectrum of the bulk protein in the steady state is obtained. This is shown in Figure 2. The frequencies of the amide I, 11, and I11 vibrations observed in the bulk spectrum of avidin are summarized in Table I. The amide I bands fall a t 1668 and 1630 cm-' , and they are attributed to the a-helix and P-sheet conformations, respectively8 (Figure 2 A ) . In the expanded amide I11 region (Figure 2 B ) , bands are observed a t 1307 and 1275 ( a helix)7 and 1250 cm-' (P-sheet).'
Difference spectra of biotin in solution a t two different pHs (5.00 and 7.40) are reported in Figure 3 together with the spectrum of biotin in KBr pellet. It was not possible to record the spectrum of biotin at p H < 5 because it precipitates. Hence the spectrum of the molecule in the solid state was recorded t o know the structure of the completely neutralized form. Table I1 summarizes the main frequencies observed, along with their assignments. Since the pKa of biotin is 4.66 (unpublished results), a t p H 5 this molecule is present partially in the neutralized and partially in the ionized form. Comparison of the spectrum of biotin a t p H 5 with that in the solid state makes it possible to correctly distinguish the absorption bands of the two different forms. By comparing these two spectra, we verified that the bands of "free" and "hydrogen-bonded'' carboxylic C = O stretching" fall a t 1700 and 1650 cm-', respectively. The band a t 1650 cm-' is due to the formation of hydrogen bonding between the COOH of different molecules." This may lead to the formation of dimers, which may in turn explain the water insolubility of the neutralized biotin. In these spectra the amide I and amide I11 bands occur a t 1680 and 1390 cm-' , respectively (Table I1 and Figure 3 ) . T h e spectrum of biotin a t p H 7.40 shows only the bands of the carboxylate group a t 1545 and 1408 cm-l ,10 in accordance with the presence of the completely ionized form a t this pH. The amide I and amide I11 bands again lie a t the same frequencies a s for p H 5, suggesting that the molecule does not change conformation with pH.
Biotin-Avidin System In Figure 4 spectra of the biotin-avidin complex ( - - - ) are compared with addition spectra of the single components (-) with biotin and avidin in the same molar ratio a s in the complex [4 : 1 ( M / M ) biotin / avidin ] .
830
BARBUCCI ET AL.
1386
1680
1480
A
1:00
cm-1
I - - -
1335
.-.Li
B
Figure 2. ( A ) Difference spectrum of avidin in solution. ( B ) Expanded amide I11 region of spectrum A.
At the first glance there are no significant differences between the two spectra within the range analyzed. However, deconvolution of these spectra [ the amide I11 region (1350-1240 cm-') was multiplied by a factor 41 brings out some differences in ab-
Table I Main Frequencies Observed in the Spectrum of Avidin in Solution Amide I
Amide I1
Amide I11
1168"m 1630b~
1540 s
1307"sh 1275"sh 1250bm
a
Bands due to a helix conformation. Bands due to /3 sheet conformation.
sorption frequencies and band intensities ( Figure 5 ) . In particular:
-A band a t 1528cm-' relative to the asymmetric stretching of COO- group" is evident in the spectrum of the complex, whereas a t this frequency only a shoulder is present in the addition spectrum. -The band a t 1650 cmpl in the sum spectrum disappears in the spectrum of the complex. -A shoulder a t 1582 cm-' is present only in the spectrum of the complex. -Some frequency shifts occur in the -CH2- vibrations range. -In the amide I11 range of the spectrum of the
ANTIGEN-ANTIBODY RECOGNITION
i
83 1
i
cm-'
Figure 3.
( A ) Difference spectrum of biotin at pH 5.00.
complex, the highest absorption occurs a t 1270 cm and a shoulder is observed a t about 1250 cm ' , whereas in the sum spectrum the highest absorption occurs a t 1250 cm-' and a weaker absorption a t 1275 cm-' (Figure 5 B ) . Bearing in mind that in the case of the sum spectrum there is no possible interaction between biotin and avidin, comparison of the two spectra in Figures
Figure 4. Difference spectrum of biotin-avidin complex (dotted line) ;addition spectrum of the single components ( solid line ) .
4 and 5 directly gives information on the interactions occurring in the biotin-avidin complex. The higher absorption a t 1528 cm-' in the spectrum of the complex with respect to that of the sum may be attributed to the drop in absorption frequency of the
Table 11 Main Frequencies Observed in the Spectra of Biotin and Their Assignments In Solution
Free carboxylic C =0 stretching Amide I Hydrogen bonded carboxylic C =0 stretching Asymmetric carboxylate C =0 stretching -CH2- deformation Symmetric carboxylate C =0 stretching Amide 111
In KBr
pH = 5.00
1700 1680 1650
1700 1680 1650 1545 1470-1430 1408 1390
1470-1430 1390
pH = 7.40
1680 1545 1470-1430 1408 1390
832
BARBUCCI ET AL.
Figure 5. ( A ) Deconvoluted difference spectrum of biotin-avidin complex (dotted line) ; deconvoluted addition spectrum of the single components (solid line). ( B ) Expanded amide I11 region of the deconvoluted difference spectrum of biotin-avidin complex (dotted line), and deconvoluted addition spectrum of the single components (solid line).
asymmetric stretching of the COO- group of complexed biotin because of a possible interaction with a n appropriate group present in the avidin macromolecule (this point will be covered later). T h e band a t 1650 cm-’ in the sum spectrum is due to the contribution of the amide I bands in both the biotin and avidin spectra (1680 cm-’ for biotin and 1668 and 1630 cm-‘ for avidin). The disappearance of this band in the spectrum of the complex may be attributed to the shift of one or more of the above-mentioned amide I absorptions to different
frequencies a s a consequence of a n interaction. As a matter of fact, a shoulder a t 1582 cm-’ appears in the spectrum of the complex, whereas it is absent in the sum spectrum. T h e small shifts observed in the -CH2-vibration range may be due to a different rearrangement of the complex with respect t o the native single components. The amide I11 region in the sum spectrum reflects the amide I11 feature of the avidin spectrum (see Table I and Figure 2B) in accordance with the fact
ANTIGEN-ANTIBODY RECOGNITION
833
I -
that the amide I11 of biotin lies a t 1390 cm-’ (Table I1 and Figure 3 B ) . Thus it may be assumed that the changes observed in this region of the spectrum of the complex are really related to biotin-avidin recognition, i.e., to biotin-avidin interactions. Biotin-l,4-Diaminobutane and Biotin-Tryptophan Systems
Biotin-l,4-&aminobutane and biotin-tryptophan systems were investigated in normal saline ( p H 7.40). Figure 6 shows the difference spectra of biotin interacting with 1,4-diaminobutane ( A ) , tryptophan ( B ) , and avidin ( C ) . Table I11 summarizes the main frequencies observed along with their assignments. In the spectrum of biotin interacting with avidin (spectrum C ) , the band due to the free amide C = O stretching ( 1680-1670-cm-’ range) completely disappears, whereas it is still present in the spectra of biotin interacting with 1,4-diaminobutane and tryptophan (spectra A and B, respectively). A drop in the “bonded” amide C = O stretching frequency is observed in the spectra of interacting biotin with respect to that of the free molecule, and the shift to lower frequencies increases from 1,4diaminobutane to tryptophan, to avidin. Since the magnitude of the drop in the amide C=O stretching frequency is related to the strength of the interaction involving this group, within the constraints of the present systems, the trend observed means that the strongest interaction involving the biotin ureido group occurs in the biotin-avidin system. This is in accordance with the idea that only the intact ureido ring is required for the strong interaction between biotin and avidin, which leads to their molecular r e ~ o g n i t i o n Other .~ interactions also occur in this system. The band a t 1545 cm-’ in the spectrum of free biotin, relative t o asymmetric COO - stretching, shifts to 1530 cm-’ in the spectrum of biotin interacting with avidin (Table 111); (this absorption occurs a t 1528 cm-’ in the spectrum of the complex). Such an interaction is supported by the spectrum
T“
i
x2
12
4 ‘r.
3
1559 cm
-’
132
Figure 6. Difference spectra of biotin interacting with ( A ) 1,4-diaminobutane,( B ) tryptophan, and ( C ) avidin.
of biotin interacting with 1,4-diaminobutane, in which the COO- asymmetric stretching of biotin occurs a t 1530 cm-’, as in the case of biotin interacting with avidin (Table 111) and we know that in
Table I11 Main Frequencies Observed in the Difference Spectra of Biotin Interacting with Avidin, Tryptophan, and 1,4-Diaminobutane and Their Assignments Asymmetric COO- Stretching
Amide I
Avidin Tryptophan 1,4-diaminobutane
“Free”
“Bonded”
“Free”
1675 1675
1576 1592 1602
1545
free Biotin: Amide I = 1680 cm-’ Asym. COO- stretch. = 1545 cm-’
“Bonded” 1530 1530
834
BARBUCCI ET AL.
the biotin-1,4 diaminobutane system only the COO. . . NH,f electrostatic interaction can occur. In fact, a t p H 7.40 the amino terminal groups of 1,4-diaminobutane are fully protonated (log kl = 10.72; log k2 = 9.44), so that they can electrostatically interact with the negatively charged carboxylate group of biotin. In the case of the biotin-tryptophan system, this interaction does not occur because tryptophan is present in the zwitterionic form a t p H 7.40 (log K1 = 9.33; log K2 = 2.35). In the spectrum of biotin interacting with tryptophan, in fact, the COOasymmetric stretching falls at 1545 cm-' (Table I11 ) , as in the case of free biotin.
CONCLUSION T h e present results confirm that interactions involving protein molecules can be recognized by F T I R / A T R spectroscopy in aqueous solution if the molecular vibrations of the groups involved are ir active. In the biotin-avidin complex, the interaction involving the COO- group of the biotin molecule had not been discovered up t o now. One of us ( C R ) thanks the EN1 for a scholarship.
REFERENCES 1. Green, N. M. (1963) Biochem. J . 89, 585-591. 2. Honzatko, R. B. & Williams, R. W. (1982) Biochemistry 21,6201-6205. 3. Bayer, E. A. & Wilchek, M. (1980) in Methods of Biochemical Analysis, Vol. 26, Glick, D., Ed., John Wiley & Sons, New York, pp. 1-45. 4. Chittur, K. K., Fink, D. J., Leininger, R. I. & Hutson, T. B. ( 1986) J . Colloid Interface Sci. 111,419-433. 5. Blas, W. E. & Helsey, G. W. (1981) Deconvolution of Absorption Spectra. Academic Press, New York. 6. Kauppimen, J. K., Moffat, D. J., Mantsch, H. H. & Cameron, D. G. (1981) Appl. Spectrosc. 35,271-273. 7. Perkin-Elmer Corporation ( 1985 ) Instructions Infrared Data System, CDS-3 Application Program Rev. D. 8. Jakobsen, R. J. & Wasacz, M. ( 1987) Proteins at Interfaces. Physicochemical and Biochemical Studies, ACS Symposium Series 343, Brash, J. L. & Horbett, T. A., Eds., American Chemical Society Publ., Washington, pp. 339-361. 9. Korpela, J . (1984) Rev. Art. Med. Biol. 62, 5-26. 10. Bellamy, L. J. (1975) The Infrared Spectra of Complex Molecules, Vol. 1, Chapman and Hall, London. 11. Gompper, R. & Herlinger, H. (1956) Chem. Ber. 89, 2825-2833.
Received July 11, 1990 Accepted February 8, 1991
’Dipartimentodi Chimica, Piano dei Mantellini, 44 53100 Siena, and ’SCLAVO S.p.A., Via Fiorentina, 1 53100 Siena, Italy
SYNOPSIS
Biotin-avidin recognition is studied by Fourier transform ir spectroscopy /attenuated total reflection (FTIR/ ATR) under physiological conditions. The ureido portion of biotin is confirmed to be involved in the interaction with avidin, as previously found, but when the biotin-avidin complex forms, an electrostatic interaction occurs between the carboxylate group of the biotin molecule and the protonated aminic end group of the avidin amino acid side chains. Comparison of the biotin-avidin system with the biotin-1,4-diaminobutane and biotin-tryptophan systems confirms these findings.
0
INTRODUCTION
II
It is known that avidin from the whites of hen eggs is a tetramer composed of 15700 M , monomers; each subunit of avidin binds one molecule of D-biotin with a n affinity (pKd 15) among the highest of all associations between naturally occurring ligands and biological macromolecules.’ Binding studies of biotin analogues have shown that the main interaction of the biotin molecule with avidin involves only the ureido portion of the ligand.’.3 In addition, the property of D-biotin to protect tryptophan residues from oxidizing agents suggests the proximity of indole side chains to the binding pocket for biotin.’ In the present study we investigated the biotinavidin system by fourier transform attenuated total reflection ir spectroscopy with the aim of -verifying the “performance” of this spectroscopic technique in analyzing interacting biological systems, and -identifying the chemical groups involved in the interactions of this antigen-antibody recognition. Riopolymer?. Vol. 31, 827-834 (1991) (c, 1991 John Wiley & Sons, Inc.
CCC 0006-3525/91/070827-08504.00
HOOC(CH,),-HC,
,CH2 S
Scheme 1. Structure of biotin.
EXPERIMENTAL Materials
+
-Lyophilized crystalline D ( ) biotin was used a s received from Merck. -Avidin was used a s received from Societii P r o dotti Antibiotici. -L-Tryptophan amino acid was used as received from Sigma Chemical Company and 1,4-diaminobutane as received from Aldrich Chemical Company. Each protein solution was prepared by dissolving the powder in a phosphate buffer solution, and the p H was adjusted by adding small amounts of aqueous HC1 or NaOH solutions. The solution concentrations were 1.5 10-3M for biotin, 1.5 10 3 M for av-
-
-
827
828
BARBUCCI ET AL.
idin, 1.0.10-2M for tryptophan, and 1.0. for 174-diaminobutane. The biotin-avidin complex solution was obtained by mixing the two components in the ratio 4 : 1 ( M / M) biotin/avidin. The biotin-tryptophan and biotin-1,4-diaminobutane systems were obtained by mixing biotin with an excess of tryptophan or 1,4-diaminobutane. Instrumentation
The spectra were obtained with a Perkin-Elmer Fourier transform ir (FTIR) spectrometer M 1800 operating between 3000 and 900 cm-’ . An MCT detector was used and the apparatus purged with dry nitrogen. Typically 300 scans at a resolution of 2.0 cm-’ were averaged and the spectra stored on floppy disks. The frequency scale was internally calibrated with a reference He-Ne laser to an accuracy of 0.01 cm-’ . A Barnes microcircle cell for liquid with a germanium crystal was used to record the spectra in solution. Spectral Processing
All the spectra in solution were taken in the single beam mode. The sample spectrum was obtained subtracting the saline solution spectrum from the sample solution spectrum. The scale factor for saline solution subtraction was chosen so that the spectral region between 2000 and 1700 cm-’ was flat. Biotin and Biotin-Tryptophan/ 7,4-Diaminobutane Systems. The difference spectra for these species in
solution were obtained directly by subtracting the spectrum of the saline solution from that of the sample solution. Avidin and Biotin-Avidin Complex. When a protein
solution is introduced into the ATR cell, there is immediate protein adsorption on the surface of the internal reflection element, so that the spectrum of the sample solution consists of two contributions: that of the adsorbed protein and that of the “bulk” protein. The term “bulk” protein refers to the protein in solution but only of layer having a thickness equal to the penetration depth of the ir beam. In order to obtain the spectrum of the bulk protein, the spectrum obtained has to be corrected for the contribution of the adsorbed protein. The spectra of the protein solutions ( avidin or biotin-avidin complex) were therefore recorded after different periods of exposure (adsorption experiment). By sub-
tracting the spectrum of the saline solution from those of the protein solution, the spectra of the (bulk adsorbed) protein at different times were obtained. When a constant spectrum was obtained, the protein solution was replaced with fresh saline solution and the spectra were collected until a constant spectrum was again achieved (desorption experiment). This spectrum represented the adsorbed protein. Subtraction of this spectrum from the one collected at the end of the adsorption experiment gave the spectrum of the bulk protein.
+
+
Spectrum “Sum” (Biotin Avidin). The addition spectrum was obtained by collecting the spectra of biotin and avidin in solution at the same molar concentrations as in the complex, and then summing the two. Spectra of Biotin Interacting with Avidin, Tryptophan, and l,4-Diaminobutane. The difference
spectra of the interacting biotin were obtained by subtracting the spectra of the native avidin, tryptophan, or 1,4-diaminobutane from those of the corresponding systems involving biotin. The criterion adopted was to minimize a pure band of the component to be subtracted. Spectral Enhancement. In order to improve the ob-
servability of the overlapping bands, mathematical resolution enhancement was performed by a spectral deconvolution process that is like the Fourier selfdeconvolution, except that the mathematical operations are performed in the spectral domain rather in the Fourier domain. The spectral deconvolution process moves intensity from the outer wings of a band into the center of the band, thus reducing its effective half width and improving its o b s e ~ a b i l i t y . ~ The quality of the deconvolution procedure is controlled by two variables, namely half-band width of the Lorentzian line used for deconvolution and the resolution enhancement achieved. 536
RESULTS AND DISCUSSION Avidin
The spectra of the protein in normal saline ( p H 7.40) collected after different periods of adsorption are reported in Figure 1. The ratio of band height at 1540 and 1528 cm-’ (correlated to the amount of the protein) increases from A (0.93) to B (0.97), to C (1.02), whereas it remains unchanged from C to D ( 1.02). Thus spectra C and D can be regarded as relative to the protein
ANTIGEN-ANTIBODY RECOGNITION
829
The higher intensity of the bands of the @sheet conformation with respect t o the a-helix confirms that in physiological solution a higher percentage of the amino acids in avidin participate in P-sheet conformation than in a-helix conformation. This agrees with the data reported in the literature ( 10% a-helix, 55% &sheet) .399
Biotin
t
--Wd o i l !
i
i
I
I
1653
I
cm'
I
I
I
I
I'
0 0
i-153
Figure 1. Difference spectra of avidin (bulk + adsorbed protein ) collected after different periods of adsorption: ( A ) 1 min, ( B ) 1 h, ( C ) 3 h, and ( D ) 24 h.
in the steady state. These protein spectra consist of the contribution of both the adsorbed and bulk protein and the changes observed within the first 3 h of exposure are presumably related to diffusion and adsorption p r o c e ~ s e s .When ~ the steady state is reached the spectrum of the adsorbed protein may be collected (see experimental section). By subtracting this spectrum from the spectrum C ( o r D ) , the spectrum of the bulk protein in the steady state is obtained. This is shown in Figure 2. The frequencies of the amide I, 11, and I11 vibrations observed in the bulk spectrum of avidin are summarized in Table I. The amide I bands fall a t 1668 and 1630 cm-' , and they are attributed to the a-helix and P-sheet conformations, respectively8 (Figure 2 A ) . In the expanded amide I11 region (Figure 2 B ) , bands are observed a t 1307 and 1275 ( a helix)7 and 1250 cm-' (P-sheet).'
Difference spectra of biotin in solution a t two different pHs (5.00 and 7.40) are reported in Figure 3 together with the spectrum of biotin in KBr pellet. It was not possible to record the spectrum of biotin at p H < 5 because it precipitates. Hence the spectrum of the molecule in the solid state was recorded t o know the structure of the completely neutralized form. Table I1 summarizes the main frequencies observed, along with their assignments. Since the pKa of biotin is 4.66 (unpublished results), a t p H 5 this molecule is present partially in the neutralized and partially in the ionized form. Comparison of the spectrum of biotin a t p H 5 with that in the solid state makes it possible to correctly distinguish the absorption bands of the two different forms. By comparing these two spectra, we verified that the bands of "free" and "hydrogen-bonded'' carboxylic C = O stretching" fall a t 1700 and 1650 cm-', respectively. The band a t 1650 cm-' is due to the formation of hydrogen bonding between the COOH of different molecules." This may lead to the formation of dimers, which may in turn explain the water insolubility of the neutralized biotin. In these spectra the amide I and amide I11 bands occur a t 1680 and 1390 cm-' , respectively (Table I1 and Figure 3 ) . T h e spectrum of biotin a t p H 7.40 shows only the bands of the carboxylate group a t 1545 and 1408 cm-l ,10 in accordance with the presence of the completely ionized form a t this pH. The amide I and amide I11 bands again lie a t the same frequencies a s for p H 5, suggesting that the molecule does not change conformation with pH.
Biotin-Avidin System In Figure 4 spectra of the biotin-avidin complex ( - - - ) are compared with addition spectra of the single components (-) with biotin and avidin in the same molar ratio a s in the complex [4 : 1 ( M / M ) biotin / avidin ] .
830
BARBUCCI ET AL.
1386
1680
1480
A
1:00
cm-1
I - - -
1335
.-.Li
B
Figure 2. ( A ) Difference spectrum of avidin in solution. ( B ) Expanded amide I11 region of spectrum A.
At the first glance there are no significant differences between the two spectra within the range analyzed. However, deconvolution of these spectra [ the amide I11 region (1350-1240 cm-') was multiplied by a factor 41 brings out some differences in ab-
Table I Main Frequencies Observed in the Spectrum of Avidin in Solution Amide I
Amide I1
Amide I11
1168"m 1630b~
1540 s
1307"sh 1275"sh 1250bm
a
Bands due to a helix conformation. Bands due to /3 sheet conformation.
sorption frequencies and band intensities ( Figure 5 ) . In particular:
-A band a t 1528cm-' relative to the asymmetric stretching of COO- group" is evident in the spectrum of the complex, whereas a t this frequency only a shoulder is present in the addition spectrum. -The band a t 1650 cmpl in the sum spectrum disappears in the spectrum of the complex. -A shoulder a t 1582 cm-' is present only in the spectrum of the complex. -Some frequency shifts occur in the -CH2- vibrations range. -In the amide I11 range of the spectrum of the
ANTIGEN-ANTIBODY RECOGNITION
i
83 1
i
cm-'
Figure 3.
( A ) Difference spectrum of biotin at pH 5.00.
complex, the highest absorption occurs a t 1270 cm and a shoulder is observed a t about 1250 cm ' , whereas in the sum spectrum the highest absorption occurs a t 1250 cm-' and a weaker absorption a t 1275 cm-' (Figure 5 B ) . Bearing in mind that in the case of the sum spectrum there is no possible interaction between biotin and avidin, comparison of the two spectra in Figures
Figure 4. Difference spectrum of biotin-avidin complex (dotted line) ;addition spectrum of the single components ( solid line ) .
4 and 5 directly gives information on the interactions occurring in the biotin-avidin complex. The higher absorption a t 1528 cm-' in the spectrum of the complex with respect to that of the sum may be attributed to the drop in absorption frequency of the
Table 11 Main Frequencies Observed in the Spectra of Biotin and Their Assignments In Solution
Free carboxylic C =0 stretching Amide I Hydrogen bonded carboxylic C =0 stretching Asymmetric carboxylate C =0 stretching -CH2- deformation Symmetric carboxylate C =0 stretching Amide 111
In KBr
pH = 5.00
1700 1680 1650
1700 1680 1650 1545 1470-1430 1408 1390
1470-1430 1390
pH = 7.40
1680 1545 1470-1430 1408 1390
832
BARBUCCI ET AL.
Figure 5. ( A ) Deconvoluted difference spectrum of biotin-avidin complex (dotted line) ; deconvoluted addition spectrum of the single components (solid line). ( B ) Expanded amide I11 region of the deconvoluted difference spectrum of biotin-avidin complex (dotted line), and deconvoluted addition spectrum of the single components (solid line).
asymmetric stretching of the COO- group of complexed biotin because of a possible interaction with a n appropriate group present in the avidin macromolecule (this point will be covered later). T h e band a t 1650 cm-’ in the sum spectrum is due to the contribution of the amide I bands in both the biotin and avidin spectra (1680 cm-’ for biotin and 1668 and 1630 cm-‘ for avidin). The disappearance of this band in the spectrum of the complex may be attributed to the shift of one or more of the above-mentioned amide I absorptions to different
frequencies a s a consequence of a n interaction. As a matter of fact, a shoulder a t 1582 cm-’ appears in the spectrum of the complex, whereas it is absent in the sum spectrum. T h e small shifts observed in the -CH2-vibration range may be due to a different rearrangement of the complex with respect t o the native single components. The amide I11 region in the sum spectrum reflects the amide I11 feature of the avidin spectrum (see Table I and Figure 2B) in accordance with the fact
ANTIGEN-ANTIBODY RECOGNITION
833
I -
that the amide I11 of biotin lies a t 1390 cm-’ (Table I1 and Figure 3 B ) . Thus it may be assumed that the changes observed in this region of the spectrum of the complex are really related to biotin-avidin recognition, i.e., to biotin-avidin interactions. Biotin-l,4-Diaminobutane and Biotin-Tryptophan Systems
Biotin-l,4-&aminobutane and biotin-tryptophan systems were investigated in normal saline ( p H 7.40). Figure 6 shows the difference spectra of biotin interacting with 1,4-diaminobutane ( A ) , tryptophan ( B ) , and avidin ( C ) . Table I11 summarizes the main frequencies observed along with their assignments. In the spectrum of biotin interacting with avidin (spectrum C ) , the band due to the free amide C = O stretching ( 1680-1670-cm-’ range) completely disappears, whereas it is still present in the spectra of biotin interacting with 1,4-diaminobutane and tryptophan (spectra A and B, respectively). A drop in the “bonded” amide C = O stretching frequency is observed in the spectra of interacting biotin with respect to that of the free molecule, and the shift to lower frequencies increases from 1,4diaminobutane to tryptophan, to avidin. Since the magnitude of the drop in the amide C=O stretching frequency is related to the strength of the interaction involving this group, within the constraints of the present systems, the trend observed means that the strongest interaction involving the biotin ureido group occurs in the biotin-avidin system. This is in accordance with the idea that only the intact ureido ring is required for the strong interaction between biotin and avidin, which leads to their molecular r e ~ o g n i t i o n Other .~ interactions also occur in this system. The band a t 1545 cm-’ in the spectrum of free biotin, relative t o asymmetric COO - stretching, shifts to 1530 cm-’ in the spectrum of biotin interacting with avidin (Table 111); (this absorption occurs a t 1528 cm-’ in the spectrum of the complex). Such an interaction is supported by the spectrum
T“
i
x2
12
4 ‘r.
3
1559 cm
-’
132
Figure 6. Difference spectra of biotin interacting with ( A ) 1,4-diaminobutane,( B ) tryptophan, and ( C ) avidin.
of biotin interacting with 1,4-diaminobutane, in which the COO- asymmetric stretching of biotin occurs a t 1530 cm-’, as in the case of biotin interacting with avidin (Table 111) and we know that in
Table I11 Main Frequencies Observed in the Difference Spectra of Biotin Interacting with Avidin, Tryptophan, and 1,4-Diaminobutane and Their Assignments Asymmetric COO- Stretching
Amide I
Avidin Tryptophan 1,4-diaminobutane
“Free”
“Bonded”
“Free”
1675 1675
1576 1592 1602
1545
free Biotin: Amide I = 1680 cm-’ Asym. COO- stretch. = 1545 cm-’
“Bonded” 1530 1530
834
BARBUCCI ET AL.
the biotin-1,4 diaminobutane system only the COO. . . NH,f electrostatic interaction can occur. In fact, a t p H 7.40 the amino terminal groups of 1,4-diaminobutane are fully protonated (log kl = 10.72; log k2 = 9.44), so that they can electrostatically interact with the negatively charged carboxylate group of biotin. In the case of the biotin-tryptophan system, this interaction does not occur because tryptophan is present in the zwitterionic form a t p H 7.40 (log K1 = 9.33; log K2 = 2.35). In the spectrum of biotin interacting with tryptophan, in fact, the COOasymmetric stretching falls at 1545 cm-' (Table I11 ) , as in the case of free biotin.
CONCLUSION T h e present results confirm that interactions involving protein molecules can be recognized by F T I R / A T R spectroscopy in aqueous solution if the molecular vibrations of the groups involved are ir active. In the biotin-avidin complex, the interaction involving the COO- group of the biotin molecule had not been discovered up t o now. One of us ( C R ) thanks the EN1 for a scholarship.
REFERENCES 1. Green, N. M. (1963) Biochem. J . 89, 585-591. 2. Honzatko, R. B. & Williams, R. W. (1982) Biochemistry 21,6201-6205. 3. Bayer, E. A. & Wilchek, M. (1980) in Methods of Biochemical Analysis, Vol. 26, Glick, D., Ed., John Wiley & Sons, New York, pp. 1-45. 4. Chittur, K. K., Fink, D. J., Leininger, R. I. & Hutson, T. B. ( 1986) J . Colloid Interface Sci. 111,419-433. 5. Blas, W. E. & Helsey, G. W. (1981) Deconvolution of Absorption Spectra. Academic Press, New York. 6. Kauppimen, J. K., Moffat, D. J., Mantsch, H. H. & Cameron, D. G. (1981) Appl. Spectrosc. 35,271-273. 7. Perkin-Elmer Corporation ( 1985 ) Instructions Infrared Data System, CDS-3 Application Program Rev. D. 8. Jakobsen, R. J. & Wasacz, M. ( 1987) Proteins at Interfaces. Physicochemical and Biochemical Studies, ACS Symposium Series 343, Brash, J. L. & Horbett, T. A., Eds., American Chemical Society Publ., Washington, pp. 339-361. 9. Korpela, J . (1984) Rev. Art. Med. Biol. 62, 5-26. 10. Bellamy, L. J. (1975) The Infrared Spectra of Complex Molecules, Vol. 1, Chapman and Hall, London. 11. Gompper, R. & Herlinger, H. (1956) Chem. Ber. 89, 2825-2833.
Received July 11, 1990 Accepted February 8, 1991
