203

JOURNAL OF BIOSCIENCE AND BIOENGINEERING 2005, The Society for Biotechnology, Japan
Vol. 100, No. 2, 203206. 2005
DOI: 10.1263/jbb.100.203
Effect of -Irradiation on the Molecular Properties
of Bovine Serum Albumin
Mohamed H. Gaber
1
Biophysics Department, Faculty of Science, Cairo University, Giza, Egypt
1
Received 20 September 2004/Accepted 28 April 2005
To investigate the effect of oxygen radicals on the molecular properties of bovine serum albu-
min (BSA), the secondary and tertiary structures, molecular weight and optical anisotropy of
BSA were examined after the irradiation of the protein at various doses. -Irradiation of the pro-
tein solution caused the disruption of the ordered structure of protein molecules as well as degra-
dation, cross-linking and aggregation of polypeptide chains. Fluorescence spectroscopy indicated
that irradiation quenched the emission intensity excited at 280 nm. Fourier transform infrared
spectroscopy (FTIR) indicated that irradiation caused transformation from -turns into -sheets.
A light scattering study showed that increasing the radiation dose decreased the molecular weight
of the protein. Optical anisotropy data showed that radiation changed the ordered structure of
the protein. Ultraviolet absorption spectroscopy indicated that fragmentation and aggregation
might occur in response to radiation exposure.
[Key words: oxygen radicals, bovine serum albumin, molecular properties, emission intensity, laser light
scattering, infrared absorption]
One of the tasks in radiation is to develop tests that in-
dicate exposure to natural or artificial ionizing radiation
fields. Some of these tests use biological indicators (bio-
dosimeters), which correlate the exposure level with the
biological effect.
Recent progress in molecular biology has revealed pre-
cise molecular mechanisms for biological reactions. The
biological effects of irradiation on mammalian cells and the
entire body have been studied at the molecular level. Ioniz-
ing radiation influences the biological responses mainly
through a reaction with intracellular or intercellular water,
which generates reactive oxygen. Reactive oxygen subse-
quently impairs DNA, or induces conformational changes in
cellular proteins (1).
The chemical changes that irradiation causes in biopoly-
mers, such as proteins, are fragmentation, cross-linking, ag-
gregation, and oxidation by oxygen radicals generated in the
radiolysis of water (2, 3). Assuming that these changes oc-
cur simultaneously, their rates depend on the chemical na-
ture of the protein, its physical state, and the irradiation con-
ditions (4). The effect of -radiation on protein conformation
appears to depend on several factors such as protein concen-
tration, the presence of oxygen and the quaternary structure
of proteins. Hydroxy radicals and superoxide anion radicals
generated by radiation modify the primary structure of pro-
teins, which results in the distortion of the secondary and
tertiary structures.
Generally, radiation causes irreversible changes at the
molecular level by the breakage of the covalent bonds of
polypeptide chains. Exposure of proteins to oxygen radicals
results in non-random and random fragmentations (5). The
protein fragmentation in aqueous solutions is affected by the
local conformation of a particular amino acid in the protein,
its accessibility to water radiolysis products, and the pri-
mary amino acid sequence (6). In a previous work (7), it has
been reported that bovine serum albumin (BSA) was cleaved
by the oxidative destruction of proline residues, yielding
specific protein fragments. Also, there have been reports on
the aggregation and cross-linking of proteins by irradiation
(8, 9). Covalent cross-linkages are formed between free
amino acids and proteins, and between peptides and pro-
teins in solution after irradiation (2).
A radiation-induced conformation of the protein structure
is observed in a number of protein systems by measuring
the changes in the molecular properties of proteins. To fur-
ther investigate the effect of oxygen radicals on the protein
structure, the radiation effect on the secondary and tertiary
structures, molecular weight and optical anisotropy of BSA
is determined in this study.
MATERIALS AND METHODS
Materials BSA was purchased from Sigma Chemical (St.
Louis, MO, USA) and used without further purification. Protein
concentration was determined by measuring the optical density at
280 nm. Fluorescence and IR measurements were carried out using
phosphate-buffered saline (PBS) filtered through a 0.22-m mem-
brane filter.
Sample irradiation Three-milliliter solutions of proteins in
10 mM phosphate buffer (pH7.0) were placed in glass vials and
irradiated at room temperature using
137
Cs -rays, at the National
Institute of Standards (Cairo, Egypt). Protein solutions were di-
e-mail: Liposome32@hotmail.com
phone: +20-202-4186754 fax: +20-202-5727556
GABER J. BIOSCI. BIOENG., 204
luted to a suitable concentration with PBS buffer before irradia-
tion. The radiation doses were 0, 0.5, 1, and 5 KGy. The protein
concentration was 0.4%.
Fluorescence spectroscopy The fluorescence emission inten-
sity of the protein solutions irradiated was measured using a spec-
trofluorometer (RF-1501; Shimadzu, Kyoto). The protein solutions
irradiated were excited at 280 nm and the emission spectra were
recorded from 300 to 450 nm.
Fourier transform infrared spectroscopy (FTIR) A film
with a small amount of protein sample (~2 l) was deposited be-
tween two disks of potassium bromide, avoiding the presence of
air. For each sample, the spectra were recorded three times with
16 scans from 3000 to 1000 cm
1
on an FTIR spectrometer (Jasco
V570; Easton, MD, USA).
UV absorption spectroscopy UV optical spectra were re-
corded at room temperature (22C) in a 1-cm cell in a -20 spectro-
photometer (Perkin-Elmer, Boston, MA, USA) in the wavelength
region 200300 nm. The protein solution was diluted with 10 mM
saline buffer (pH7.0) to achieve the desired protein concentration.
The reported UV spectra were the average of five scans and were
smoothed by a polynomial curve fitting program. The error in the
reading was less than 8% (standard error estimation).
Laser light scattering analysis A slightly modified home-
made light scattering spectrometer with a helium neon laser (630
nm wavelength and 50 mW output power) was used as the light
source. The incident beam was vertically polarized with respect to
the scattering plane. The detector was a YAg-100 A photodiode
(EG&G Judson, Gaithersburg, MD, USA). The device had an ac-
tive area of 5.1 mm
2
and when struck by a photon of sufficient
energy, an electron-hole pair was created in the photodiode. Cor-
rect biasing of the diode generated a current from this event. The
photocurrent was converted to a voltage by a transimpedance
preamplifier, and the voltage output from the preamplifier was fed
to a sensitive digital voltmeter, where the voltage signal was dis-
played.
The molecular weight of the protein sample was calculated on
the basis of the light scattering theory (10). For a dilute macro-
molecule solution at a concentration C (g/ml) and scattering angle
, the angular dependence of the excess absolute average scattered
intensity, known as the excess Rayleigh ratio [R
VV
()], can be ap-
proximated as
(1)
where K=4
2
n
2
( )
2
/(N
A

0
4
), with N
A
, n, and
0
being Avogadros
number, the solvent refractive index, and the wavelength of light
in a vacuum, respectively, and q=(4n/
0
)sin(/2). After measur-
ing R
VV
() for a set of C and , we determined the weight-average
molar mass (M
W
) for the protein sample at different doses of radia-
tion from the Zimm plot which incorporates and C extrapolations
on a single grid (11).
To evaluate the polarization ratio for protein samples at differ-
ent doses of radiation, the intensity of the components of the scat-
tered light, the electric vector of which vibrates perpendicular and
parallel to the plane of observation, I
1
() and I
2
(), were measured
at a fixed angle, and the polarization ratio was defined as follows
(12):
() = (2)
The polarization ratio is a measure of the optical anisotropy of
the molecule.
RESULTS
Figure 1 shows the fluorescence emission intensity for
BSA when excited at 280 nm. The emission intensity was
measured at different doses of -radiation. The results show
that increasing of the radiation quenched the emission inten-
sity of the protein.
UV absorption spectra show the conformational change
in the secondary structure of proteins, particularly in the
case of a change in the local environment of the ordered
structure of a polypeptide chain. The UV spectra of BSA so-
lution irradiated at various doses were obtained (Fig. 2).
The data show a decrease in absorption intensity at a low
dose of radiation (0.5 KGy) and an increase in absorption
intensity at high doses of radiation (1 and 5 KGy). There
was a negligible shift in the absorption wavelength at differ-
ent doses of radiation.
Since infrared spectroscopy is a well-established tech-
nique for the analysis of the protein secondary structure, the
FTIR spectra of the BSA solution irradiated at various doses
were obtained (Fig. 3). It is clear from Fig. 3 that exposure
of the protein samples to radiation doses of 1 and 5 KGy
caused a shift in the main band position which is located at
KC
R
VV
( )
-------------------
1
M
W
------------ 1
1
3
----- R
g
2

Z
q
2
+


n C
I
2
( )
I
1
( )
---------------
FIG. 1. Fluorescence emission spectra of BSA irradiated at an ex-
citation wavelength of 280 nm.
FIG. 2. UV absorption spectra of BSA irradiated.
MOLECULAR CHANGES IN SERUM BY RADIATION VOL. 100, 2005 205
1640 cm
1
to a lower wave number. However, exposure to
the 0.5-KGy radiation dose caused a minor change in the
main band position of the FTIR spectrum.
Light scattering analysis was used to elucidate the effect
of radiation on the molecular weight of the protein. Figure 4
shows a marked decrease in the molecular weight of BSA
when exposed to -radiation at 1 and 5 KGy.
To further investigate the damaging effect of radiation on
the protein structure and integrity, the light scattering tech-
nique was used to measure the optical anisotropy of the
protein sample at the different doses of radiation. Figure 5
shows the optical anisotropy of BSA at various doses of
radiation. The data show a considerable increase in the opti-
cal anisotropy of the protein sample when exposed to -radi-
ation at 0.5 and 5 KGy radiation dose. However, no remark-
able changes obtained when exposed to 1 KGy dose.
DISCUSSION
Radiation treatments of biological materials have been
applied to food products and radiation causes irreversible
changes in protein conformation at the molecular level. The
chemical changes are fragmentation, cross-linking, and ag-
gregation by oxygen radicals generated in the radiolysis of
water. Therefore, oxygen radicals modify the structure of
proteins, which results in changes in physicochemical prop-
erties (13).
To determine the changes in the molecular properties of
BSA protein solution due to irradiation, fluorescence emis-
sion intensity was measured. When the protein was excited
at 280 nm, which excited the tryptophan and tyrosine resi-
dues, the tertiary structure of the protein was reflected. Fig-
ure 1 shows that -irradiation caused a decrease in the emis-
sion intensity of BSA due to the change in the local environ-
ment around tryptophan and tyrosine residues (14). Increas-
ing the dose of radiation quenched the emission intensity of
the bovine serum protein.
The changes in the UV spectra of BSA due to radiation
were mainly due to the cleavage of the covalent bonds of
protein molecules and the formation of aggregates. How-
ever, the decrease in absorption intensity obtained at a low
dose could be attributed to the change in the local environ-
ment of the ordered structure of polypeptide chains. The UV
results support the finding that -irradiation breaks covalent
bonds easily and disrupts the ordered structure of proteins,
resulting in unnatural protein products (13, 14).
Infrared spectroscopy is well established as the method
for the analysis of the protein secondary structure in solu-
tion. The singular advantage of FTIR over other techniques
is that spectra can be obtained for proteins in a wide range
of environments (15).
There is a wealth of information that can be used to de-
rive structural information by analyzing the shape and posi-
tion of bands in the amide I region of the spectrum. The
presence of a number of amide I band frequencies has been
correlated with the presence of -helical, antiparallel, and
parallel -sheets and random coil structures (16, 17). -Sheet
vibrations have been shown to absorb between 1640 and
1620 cm
1
(1820). -Irradiation of BSA at 1- and 5-KGy
doses caused a shift in the peak position which had been
centered at 1640 cm
1
, indicating the formation of -sheets.
Infrared absorbances are greatly affected by the local mi-
FIG. 3. FTIR absorption spectra of BSA irradiated.
FIG. 4. Effect of -irradiation on the molecular weight (KDa) of
BSA as determined by the angular light scattering technique.
FIG. 5. Effect of -irradiation on the optical anisotropy of BSA as
determined by the depolarized light scattering technique.
GABER J. BIOSCI. BIOENG., 206
croenvironment of the different structural groups.
Regarding the radiation damage to proteins, two types of
damage are observed: fragmentation and aggregation (4).
Light scattering data of BSA (73 KDa) showed that -irradi-
ation above 0.5 KGy causes breakdown of the polypeptide
chain, resulting in the formation of degraded low-molecu-
lar-weight molecules. Similar results were observed in other
studies (7, 21). Le Maire et al. (21) suggested that the loca-
tions of the break points caused by aspartate transcarbamy-
lase radiation are fragile bonds in the polypeptide chain.
Schuessler and Schilling (7) proposed that proline residues
are the targets for chain scission caused by radiation. Wolf
et al. (22) reported that a peptide bond could be cleaved by
the direct oxidation of proline residues.
Proteins may be converted to high-molecular-weight ag-
gregates due to the generation of interprotein cross-linking
reactions, hydrophobic and electrostatic interactions, and the
formation of disulfide bonds (3, 21). Any amino acid radi-
cal formed within a peptide chain could cross-link with an
amino acid radical in another protein. In the high-dose range,
BSA proteins exposed to radiation underwent covalent cross-
linking. The formation of high-molecular-weight aggregates
was negligible in the low dose range and increased signifi-
cantly with higher doses (23, 24).
In conclusion, -irradiation of BSA protein solutions
caused the disruption of the ordered structure of protein
molecules as well as degradation, cross-linking, and aggre-
gation of the polypeptide chains due to oxygen radicals gen-
erated by the radiolysis of water, and altered the secondary
structure, tertiary structure, and molecular weight profiles
of BSA.
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