J. Clarkson*, C. Sudworth, S.I. Masca, D.N. Batchelder & D.A. Smith, “Ultraviolet resonance Raman study of the avidin biotin complex”, J. Raman Spectrosc., 31, 373-375, 2000.

1. JOURNAL OF RAMAN SPECTROSCOPY
J. Raman Spectrosc. 31, 373–375 (2000)
Ultraviolet resonance Raman study of the avidin
biotin complex
John Clarkson,* Caroline Sudworth, Sergiu I. Masca, David N. Batchelder and D. Alastair Smith
Department of Physics and Astronomy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK
An ultraviolet resonance Raman, UVRR, spectroscopic study of avidin and avidin/biotin, at pH 7.5, performed
with 244 nm excitation, resolves spectral contributions from the tyrosine 33 and tryptophan residues. The
UVRR difference spectrum of holo avidin minus apo avidin, reveals details of the changes to avidin’s
tyrosine 33. No change in the hydrogen bonding state of tyrosine 33 is detected, but a change to a more
hydrophobic environment is indicated upon addition of biotin. Tryptophan bands dominate the difference
spectrum, with changes corresponding to the movement of tryptophan tortional angles being observed. The
increase in tryptophan UVRR bands upon addition of biotin indicates a change to a more hydrophobic
environment, highlighting the importance of hydrophobic interactions in the observed strong avidin/biotin
binding. Copyright © 2000 John Wiley & Sons, Ltd.
INTRODUCTION
Biotin forms one of the strongest known protein substrate
complexes with avidin, a hen egg white protein. This very
high binding affinity has resulted in the complex being
exploited in numerous biotechnological applications.1,2
Hydrogen bonding to the ureido part of biotin is known to
play an important role in the interaction. Chemical modi-
fication of the single tyrosine residue, Tyr33, results in the
complete loss of biotin binding, as does chemical modifi-
cation of the tryptophan side groups, Trp 70 or Trp 110.3,4
The fluorescence of avidin Trp residues is also modified
by biotin binding, showing a 34% reduction in quantum
efficiency in the complex.5,6
Ultraviolet absorbance spec-
tral changes have been interpreted as evidence that the
Trp residues move into a more hydrophobic environment
upon biotin binding.7
Here we present the first reported results from an
ultraviolet resonance Raman (UVRR) spectroscopic study
of the binding of biotin by avidin. Previous reported
visible and near-infrared Raman studies of avidin/biotin
provided evidence of secondary structural changes to
avidin. An increase in the intensity of bands assigned to
Trp residues upon biotin binding was also observed.8–11
The UVRR study presented here, using 244 nm radiation,
provides selective enhancement of the Trp and Tyr Raman
signals.12
This enables unambiguous assignment of the
changes to Tyr33 and the Trp residues upon complex
formation, revealing further details regarding the nature
of the reorientation of the binding pocket of avidin upon
addition of biotin.
EXPERIMENTAL
Avidin and biotin where purchased from Sigma. A solu-
tion of 200 µM avidin was prepared in PBS buffer
* Correspondence to: J. Clarkson, Department of Physics and Astron-
omy, E.C. Stoner Building, University of Leeds, Leeds, LS2 9JT, UK;
e-mail: phyjcl@phys-irc.novell.leeds.ac.uk
at pH 7.5 containing 5 mM KNO3, as an internal
Raman intensity standard. Raman spectra were obtained
using 2 mW, 244 nm radiation from an intracavity
wavenumber-doubled argon ion laser (Coherent Innova
300 FReD). A Renishaw micro-Raman System 1000
spectrometer modified for use at 244 nm was used
to acquire the spectra,13
which were accumulated over
30 minutes, (60 ð 30 seconds). To avoid photodegrada-
tion the protein solution was rapidly circulated through
a quartz capillary tube, (i.d. D 0.2 mm) by a peri-
staltic pump (tubing i.d. D 0.5 mm) from a reser-
voir at 4 °C. Volumes as low as 200 µL could be used
with this apparatus. To minimize dilution of the pro-
tein solution, solid biotin, approximately 10 times in
excess of avidin, was added to one aliquot of avidin
solution, gently shaken for 5 minutes and kept on ice
for 1 hour.
RESULTS AND DISCUSSION
Figure 1(a) shows the UVRR spectrum of apo avidin,
(no biotin present), at 244 nm. (The spectral features
in Fig. 1 are assigned according to previous reports of
UVRR studies of proteins).12,14
The selective enhance-
ment of the Trp and Tyr residues is evident and the
amide I band at 1662 cm 1
is also reasonably strong. Phe
is not resonantly enhanced at this wavelength and does
not contribute to the spectra, despite avidin having seven
Phe residues. A similar result has been reported for the
UVRR spectrum of lysozyme with 244 nm excitation.15
Upon binding of biotin a number of spectral changes
occur in the Tyr and Trp intensities and peak positions,
[Fig. 1(b)], that are highlighted in the difference spec-
trum, [Fig. 1(b-a)]. Biotin does not exhibit measurable
Raman bands at 244 nm, as it is a very weak Raman
scatterer, with no electronic absorbance near 244 nm
and makes no contribution to the difference spectrum.
We shall consider the interpretation of these data in
terms of the effects on Tyr33 and the Trp residues
separately.
Copyright © 2000 John Wiley & Sons, Ltd. Received 15 September 1999
Accepted 15 October 1999

2. 374 J. CLARKSON ET AL.
Figure 1. UVRR spectrum of (a) apo avidin, (b) holo avidin (biotin
present), and (b a) the difference spectrum. The intensities of
(a) and (b) are normalized to the internal standard NO3 peak at
1047 cm 1
and the difference spectrum is scaled by a factor of 2
for clarity. The asterisk indicates cosmic ray interference.
Tyrosine 33
The derivative features in the difference spectrum (pairs
of positive and negative peaks) at 1604/1617 cm 1
and
1159/1174 cm 1
are assigned to upshifts of the Y8a and
Y9a bands, respectively. Similar features in the UVRR
difference spectra associated with structural changes in
bacteriorhodopsin, octopus rhodopsin and haemoglobin
have been reported.16–19
An intensity increase in the tryp-
tophan W1 peak is likely to contribute to the 1617 cm 1
feature in the difference spectrum. The increase in the
intensity of Y7a peak, however, suggests that the differ-
ence feature at 1617 cm 1
has a large contribution from
a shift and increase in intensity of Y8a. The Y8a peak of
tyrosine is more than twice as intense as Y7a.
The UVRR intensities and wavenumbers of Y8a, Y8b,
Y7a and Y9a Raman bands are reported to be sensitive
to hydrogen bonding and hydrophobic effects.19,20
The
noticeable increase in the intensity of Y7a in the difference
spectrum, indicates a more hydrophobic environment for
Y33 upon the addition of biotin. The other tyrosine bands
at 857 and 829 cm 1
in the difference spectrum are due to
an increase in the intensity of the Y1 Fermi doublet. This
Fermi doublet is also sensitive to the hydrogen bonding
state of the tyrosine and the ratio of the intensity of
the 852 cm 1
band to the 826 cm 1
band increases with
increasing enthalpy of hydrogen bond formation.19–22
Our
data show a ratio of 0.97 for the complex and 0.98 for
avidin alone, which suggests very little difference in the
hydrogen bonding state of Tyr33 upon biotin binding, in
agreement with previous visible and near infrared Raman
studies.8–11
The x-ray structures of the holo and apo
forms of avidin reveal that the ureido oxygen of biotin
forms a hydrogen bond with the Tyr33 of holo avidin,
with apo avidin having several water molecules in the
binding pocket, one being hydrogen bonded to Tyr33.23–25
This may account for the absence of a change in the
hydrogen bonding state of Tyr33 indicated by our data.
The displacement of water molecules from the binding
pocket and the formation of a hydrophobic and more polar
environment upon biotin binding would account for all the
changes we observe in the UVRR bands of Tyr33.
Tryptophan
The dominant contributions to the difference spectrum
can be assigned to the tryptophan residues. The W3 peak
wavenumber shows a strong correlation to the torsional
angle of tryptophan and the 1549 cm 1
peak in Fig. 1(a)
and 1(b) represents an average value of approximately 90°
of j 2,1
j for the four Trp residues in apo and holo avidin.
The difference spectrum reveals that for holo avidin there
is an apparent increase in intensity and also splitting of the
W3 peak, giving peaks at 1556 cm 1
and 1547 cm 1
. The
small negative feature at 1585 cm 1
complicates the W3
feature in the difference spectrum and is due to an upshift
in the W2 wavenumber profile. The increase in intensity
of the W3 features is due to the expulsion of water and
the formation of a hydrophobic pocket with a concomitant
decrease in local dielectric constant when biotin is bound.
The splitting of W3, revealed in the difference spectrum,
reflects the reorganization of the tryptophan residues upon
addition of biotin.
The W7 Fermi doublet also shows a noticeable increase
in intensity in the holo form. The ratio of the peak inten-
sity of the 1359 cm 1
to the 1344 cm 1
band reflects the
polar nature of the Trp environment.12,26
Apo avidin has a
1359/1344 cm 1
value of 1.08 and holo avidin 1.15. The
increase in this ratio upon addition of biotin reflects the
expulsion of water and the hydrophobic nature of biotin.
All the other Trp features in the difference spectra at
1492, 1462, 1432, 1238, 1009, 877 and 760 cm 1
are
due to increases in intensity from W4, W5, W6, W10,
W16, W7 and W18, respectively. The general increase
in intensity for the Trp UVRR bands upon addition of
biotin is much more profoundly illustrated here than in
previous visible Raman studies and indicates a change to
a more hydrophobic environment for these residues.8–11,16
The wavenumber of W17 is a marker of the degree of
hydrogen bonding of the Trp indole ring.27
No change
in wavenumber is observed, only an increase in inten-
sity, which indicates that there is no change in the aver-
age degree of hydrogen bonding of the four avidin Trp
residues, similar to our observations concerning the hydro-
gen bonding of Tyr33.
The UVRR difference spectrum presented here resolves
the Tyr33 and the Trp contributions to the binding of biotin
by avidin. Changes in the ultraviolet absorbance spectral
Copyright © 2000 John Wiley & Sons, Ltd. J. Raman Spectrosc. 31, 373–375 (2000)

3. ULTRAVIOLET RESONANCE RAMAN STUDY OF THE AVIDIN BIOTIN COMPLEX 375
properties of Tyr33 are obscured by the dominant effect
of Trp at pH 7.5.28
We have demonstrated the ability of
UVRR to give better molecular specificity, allowing for
unambiguous assignment of the Raman peaks to molecular
changes to the Tyr33 residue of avidin. The UVRR method
also allows changes in the molecular orientation and
environment of the Trp residues to be revealed in solution
measurements. Our results also indicate that there is little
change in the hydrogen bonding state of Tyr33 or the Trp
residues and provide further evidence of the importance
of hydrophobic interactions in stabilizing the avidin/biotin
complex.
REFERENCES
1. Green NM. Adv. Protein Chem. 1975; 29: 85.
2. Wilchek M, Bayer EA. Methods in Enzymology. Academic
Press: London, 1990.
3. Gitlin G, Bayer EA, Wilchek M. Biochem. J. 1990; 269: 527.
4. Gitlin G, Bayer EA, Wilchek M. Biochem. J. 1988; 250: 291.
5. Mei G, Pugliese L, Rosato N, Toma L, Bolognesi M,
Finazziagro A. J. Mol. Biol. 1994; 242: 559.
6. Kurzban GP, Gitlin G, Bayer EA, Wilchek M, Horowitz PM.
Biochemistry 1989; 28: 8537.
7. Green NM. Biochem. J. 1963; 89: 599.
8. Honzatko RB, Williams RW. Biochemistry 1982; 21: 6201.
9. Fagnano C, Fini G, Torreggiani A. J. Raman Spectrosc. 1995;
26: 991.
10. Fagnano C, Torreggiani A, Fini G. Biospectroscopy 1996; 2:
225.
11. Torreggiani A, Fini G. J. Raman Spectrosc. 1998; 29: 229.
12. Harada I, Takeuchi H. In Spectroscopy of Biological Systems,
Clark RJH, Hester RE (eds). Wiley: Wichester, 1986; 113 175.
13. Williams KPJ, Pitt GD, Batchelder DN, Kip BJ. Appl.
Spectrosc. 1994; 48: 232.
14. Hu XH, Spiro TG. Biochemistry 1997; 36: 15701.
15. Kaminaka S, Kitagawa T. Appl. Spectrosc. 1992; 46: 1804.
16. Hashimoto S, Sasaki H, Takeuchi H. J. Am. Chem. Soc. 1998;
120: 443.
17. Ames JB, Ros M, Raap J, Lugtenburg J, Mathies RA.
Biochemistry 1992; 31: 5328.
18. Hashimoto S, Takeuchi H, Nakagawa M, Tsuda M. Febs
Letters 1996; 398: 239.
19. Rodgers KR, Su C, Subramaniam S, Spiro TG. J. Am. Chem.
Soc. 1992; 114: 3697.
20. Hildebrandt PG, Copeland RA, Spiro TG, Otlewski J,
Laskowski M, Prendergast FG. Biochemistry 1988; 27:
5426.
21. Tu AT. In Spectroscopy of Biological Systems, Clark RJH,
Hester RE (eds). Wiley: Chichester, 1986; 47 112.
22. Couling VW, Foster NW, Klenerman D. J. of Raman
Spectrosc. 1997; 28: 33.
23. Pugliese L, Coda A, Malcovati M, Bolognesi M. J. Mol. Biol.
1993; 231: 698.
24. Pugliese L, Malcovati M, Coda A, Bolognesi M. J. Mol. Biol.
1994; 235: 42.
25. Livnah O, Bayer EA, Wilchek M, Sussman JL. Proc. Natl.
Acad. Sci. U.S.A. 1993; 90: 5076.
26. Harada I, Miura T, Takeuchi H. Spectrochimica Acta Part A
1986; 42: 307.
27. Miura T, Takeuchi H, Harada I. Biochemistry 1988; 27: 88.
28. Green NM. Biochem. J. 1963; 89: 609.
Copyright © 2000 John Wiley & Sons, Ltd. J. Raman Spectrosc. 31, 373–375 (2000)