Int. J. Peptide Protein Res.

20, 1982, 238-245

Pigeon egg white lysozyme

Purification, structural and enzymic characterization

J.G.GAVILANES, G . GONZALEZ DE BUITRAGO, A.,MART~NEZDEL POZO, R. PEREZCASTELLS

and R. RODRIGUEZ
Department of Biochemistry, Faculty of Chemistry, Complutense University, Madrid, Spain

Received 28 October 198 1, accepted for publication 16 March 1982

Lysozyme from pigeon egg white has been purified by ion exchange chromatogra-
phy and gel filtration. The overall yield of the purification procedure is 65%.
The specific activity of the enzyme is 15 000 units/mg. The influence of pH and
ionic strength on the lytic activity of the protein, as well as its thermal stability,
have been studied. The molecular weight, secondary structure estimation, amino
acid composition, NH2- and COOH-terminal sequence of the protein are also
reported. The pigeon enzyme has been classified as a chicken type lysozyme
(lysozyme c) according to the obtained results.
Key words: pigeon lysozyme, circular dichroism, sequence of proteins.

The lysozymes constitute a group of enzymes zymes has been valuable for interesting studies
widely distributed in nature with the common on molecular evolution and antigenic structure
feature of their lytic ability towards bacteria. (5-7). Also, the comparison of homologous
The enzyme obtained from the egg white of proteins from different species can contribute
hen has been extensively characterized. In towards the understanding of the protein
addition to that, structural and enzymic studies structure-function relationships. In the present
have been performed on many avian egg white paper we report the isolation and character-
lysozymes (1 -4). However, most of the studies ization of pigeon (Columba livia) egg white
on bird lysozyme have been done with Galli- lysozyme. Thus, the reported studies on the
formes and Anseriformes, the last one being pigeon enzyme extend the comparison of lyso-
the only order of birds comprising species zymes to the Columbiformes order.
having both a chicken type (lysozyme c) and a
goose type (lysozyme g) in their egg white
(4). The enzyme from Columbiformes has not MATERIALS AND METHODS

_ - __
Abbreviations: SDS, sodium dodecyl sulphate; u.v., obtained from b S 'Tilos farm ( h S Matas,
ultraviolet; CD, circular dichroism; PTH, phenyl- Madrid). Hen egg white lysozyme has been
thiohydantoin; t.l.c., thin-layer chromatography;g.l.c., purified as previously described (8). Bacterial
gas-liquid chromatography. cell walls from Micrococcus luteus (M.lysodeik-
238 0367-8377/82/080238-08 $02.00/0 0 1982 Munksgaard. Copenhagen
 Pigeon egg white lysozyme
ticus), horse heart cytochrome c, sperm whale the method of Lowry et al. (10) using horse
myoglobin, a-lactalbumin, insulin and carboxy- serum albumin as standard. When pure, the
peptidase Y were purchased from Sigma Co. enzyme concentration was determined spectro-
(St. Louis, MO, USA). Bovine pancreas trypsin, photometrically using an of 1.5.
pig stomach pepsin and PMSF-treated carboxy-
peptidase A were from Worthington (Freehold, A n a b tical methods
NJ, USA). 5,5'-dithiobis(2-nitrobenzoic acid) Polyacrylamide gel electrophoresis was per-
was purchased from Calbiochem (Los Angeles, formed according to the method of Panyim &
CA, USA). Sephadex G-50 and Sephadex G-75 Chalkley (1 1). SDS-polyacrylamide gel electro-
were obtained from Pharmacia (Sweden). All phoresis was carried out according to King &
other reagents were obtained from Merck Laemmli (12) in a 13-30% acrylamide gradient.
(Darmstadt, W. Germany) or Sigma Co. (St. Prior to the SDS-electrophoresis, samples were
Louis, MO, USA). Thin-layer chromatography incubated at 100" in the presence of 0.2%
polyamide layers were obtained from Schleicher (w/v) SDS and 1% (v/v) 2-mercaptoethanol.
und Schiill (F-1700) (Dassel, W. Germany), Purity of the enzyme preparations were also
checked by chromatography in a Biogel A
Atrijication of lysozyme 0.5 m column (1 x 50 cm) equilibrated in 0.1 M
Homogenization: lyophilized pigeon egg white potassium phosphate buffer pH 6.6 (1 3).
(1 5 g) was dissolved in 30 ml of 0.1 M potassium Lysozyme was eluted from the column with
phosphate buffer, pH 6.6. Undissolved material 0.8 M potassium phosphate buffer pH 6.6.
was removed by centrifugation at 27 OOOg for
15 min at 4". The pellet was reextracted twice
with 40 ml phosphate buffer. Molecular weight determination
Adsorption on Amberlite resin: Amberlite Pigeon egg white lysozyme and suitable marker
CG-50 equilibrated in 0.1 M phosphate buffer, proteins were chromatographed at 4" on a
pH 6.6, was stirred overnight at 4" with the Sephadex G-75 column (1 x 85 cm) equilibrated
clear supernatant from the former step. lOml in 0.1 M potassium phosphate buffer, pH 6.6,
resin were used at this step. The lysozyme- containing 0.02% (w/v) sodium azide. The
bound resin was washed with the equilibration elution was performed in the same buffer.
buffer until no absorbance at 280nm was Marker proteins were: horse heart cytochrome c,
detected. The enzyme was eluted from the resin mw 1 2 3 0 0 (14); dactalbumin, mw 1 4 4 0 0
with 0.8 M potassium phosphate buffer, pH 6.6, (1 5 ) ; hen egg white lysozyme, mw 14 500 (1 6);
at 4". Fractions with enzyme activity were sperm whale myoglobin, mw 1 7 8 0 0 (16);
pooled. pig stomach pepsin, mw 35 500 (17); bovine
Sephadex (2-50chromatography: The sample pancreas trypsin, mw 23 600 (14); insulin,
from the ion exchange step was applied directly mw 11 400 (18) and bovine pancreas ribo-
to a Sephadex G-50 fine column (5 x 27cm) nuclease A, mw 13500 (14). The reference
equilibrated in 0.1 M phosphate buffer. The proteins were detected in the effluents by
elution was performed with the same buffer. absorbance at 280 nm and/or enzyme activity.
Fractions with lysozyme activity were pooled
and store frozen until required. Spectroscopic studies
The absorption spectrum of the enzyme was
Enzyme assays obtained in a Cary 118 spectrophotometer at a
Lytic activity was determined at 20" by scanning speed of 0.2nm/s and auto slit
observing the decrease in the absorbance at program. Prior to the determination, the
4 5 0 n m that occurred during the lysis of a samples were routinely filtered through Milli-
suspension of dead cells of M. luteus at pH 6.2 pore (0.5pm pore diameter). Circular dichroism
and 0.1 5 M ionic strength phosphate buffer (9). spectra of the enzyme were measured in a Jobin
Yvon Mark 111 dichrograph fitted with a 250W
Protein detennination xenon lamp at a 0.2nm/s scanning speed.
Protein estimation was performed according to Millipore (0.5 pm pore diameter) filtered

239
J.G. Gavilanes et al.
solutions were studied at 0.2 mg/ml protein
concentration, using 0.05 cm and 1 cm optical
path cells in the far- and near-u.v. respectively.
The values of mean residue weight ellipticities
were calculated on the basis of 110 for the
amino acids in the enzyme, and they are
reported in terms of Omrw (degrees x cm2 x
dmol-' ). Secondary structure estimations were
performed by computer fit according to the
ellipticity reference values of Chen et al. (19).
Amino acid analysis
Lysozyme was hydrolyzed at 108" for different
lengths of time (24, 48, 72, 96 and 120h)
with 5.7 N HCl containing 0.1% (w/v) phenol,
in evacuated sealed tubes. Hydrolysates were
analyzed on a Durrum amino acid analyzer
(model D 500). For each hydrolysis time, three
separate determinations were performed. Half
cystine was determined as cysteic acid after
oxidation with performic acid at - 10" (20).
The number of total -SH groups was estimated
by reaction with 5,5'-dithiobis(2-nitrobenzoic
acid) (21) in 0.1 M Tris buffer, pH 7.5, con-
taining 1 mM EDTA and 0.3% (w/v) SDS. A
molar absorption coefficient of 1 3 600 M
cm-' at 41 2 nm for 5-thio-2-nitrobenzoate
was employed for calculation. Tryptophan was
determined spectrophotometrically in 0.1 M
NaOH (22,23).
NH2-terminal sequence
The NH, -terminal amino acid sequence was
determined on an automatic sequencer
(Beckman 890 B). All the reagents were
sequence grade (Beckman, Palo Alto, CA,
USA). Double coupling and double cleavage
at proline residues as well as double cleavage at
isoleucine residues were used to improve the
yield of the sequence. The PTH-amino acid
residues were identified by back hydrolysis
(24), two-dimensional t.1.c. on polyamide layers
( 2 5 ) and g.1.c. on a Hewlett Packard 5750 gas
chromatograph, SP 400 column.
COOH-terminal sequence
Carboxypeptidase A digestion was carried out
with native lysozyme at 37" for 15 h in 0.1 M
ammonium bicarbonate for a 20: 1 lysozymel
enzyme weight ratio. Carboxypeptidase Y
digestion was performed with the performic
acid oxidized lysozyme. The protein was dis-
solved in 0.05 M pyridine-acetate buffer pH 5.5
and incubated at 37" with carboxypeptidase Y
for different lengths of time. The lysozyme/
carboxypeptidase weight ratio was 20: 1. Ali-
quots were removed and lyophilized for amino
acid analysis. Norleucine was used as an internal
standard for quantitative determination of the
released amino acids,
RESULTS
Purification of the enzyme
Table 1 summarizes the purification procedure
for lysozyme from pigeon egg white. The
overall yield was 65% and the final purification
98-fold; the specific activity of the purified
enzyme preparation was 15 000 units/mg. The
elution profiles of the Amberlite CG-50 and
Sephadex G-50 chromatographic steps are given
in Figs. 1 and 2 respectively. After the last
purification step, the enzyme moves as a single
band on polyacrylamide gel electrophoresis in
the presence of SDS. A single peak was also
found in the Biogel A 0.5m chromatography.

TABLE 1
Purification of lysozyme from 15g lyophilized pigeon egg white

Step (mg) (units) (unitslmg) (%I

total protein total activity specific activity yield

Homogenate 750 115 000 153 100

Amberlite CC-50 150 80 500 537 70
Sephadex G-50 5 75 000 15 000 65

100 ml pigeon egg white give about 13 g lyophilized material. Homogenization of frozen egg white results in 2-3
times lower specific activity than that obtained with the lyophilized sample.

240

tt
10 20 30
fraction
FIGURE 1
Elution profie of the Amberlite CG-50 column
chromatography. (-) absorbance at 280 nm and
(- -- -) enzymic activity. Experimental conditions
are described in Materials and Methods. 1-ml fractions
were collected.
Taking into account the specific retention of
lysozyme on agarose gels (8, 13) this chromato-
graphic analysis is also considered an homo-
geneity index. Any other subsequent purifi-
cation step did not increase the specific activity
of the enzyme preparation.
Pigeon egg white lysozyme purification can
not be achieved in a single step, by chromato-
graphy in agarose gels as described for other
lysozymes (8), due to the existence of a con-
taminant protein which is not completely
resolved except after chromatography on
Amberlite CG-50 column.
The purified enzyme has a relative electro-
phoretic mobility (1 1) to the hen lysozyme of
1 .O in the absence of SDS.
The specific activity of the purified enzyme
from pigeon egg white is 2.7 times lower than
that obtained for hen lysozyme (type c). The
specific activity reported for goose type (lyso-
zyme g) is 6-10 times higher (26,27) than that
Pigeon egg white lysozyme
of pigeon lysozyme. The lysozyme contents in
the egg white from pigeon and hen are about
0.60% and 0.45%, respectively, of the total
solubilized protein, according to our homogen-
ization procedure. Lysozyme content in the egg
white reported for the goose type enzyme
(lysozyme g) is 20-23 times lower (26) than
that obtained for the protein from pigeon.
Enzyme characterization
The results of the thermal stability study are
given in Fig. 3 in which hen egg white lysozyme
is also considered for comparison. At 10min-
incubation, the pigeon enzyme shows higher
stability at acidic pH than at alkaline pH values,
in agreement to the properties of other lyso-
zymes previously described (28, 29). However,
in these conditions, the pigeon egg white
enzyme is less stable than the hen egg white
lysozyme for high temperature treatment. After
10min at 90°, pH 3.3, the pigeon enzyme has
40% of its activity whereas 80% of the hen
lysozyme activity is preserved.
The influence of the pH on the lysis of M.
luteus cell walls shows a biphasic profile (Fig.
4), with activity maxima centered at 5.0-
5.5 and 7.0-7.5. This determination has been
performed at 0.1 M ionic strength. At these
conditions, the hen lysozyme shows also a
biphasic optimum pH at approximately the
same pH values (Fig. 4). However, the activity
at the acidic maximum for the hen is double
that for the pigeon egg white enzyme. These
10 20 30 40 50 fraction
FIGURE 2
Elution profile of the chromatography on Sephadex
G-50 column. )-( absorbance at 280nm and
(- -- -) enzymic activity. Experimental conditions
are as described in Methods. 4-ml fractions were
collected.
24 1
J.G. Gavilanes et af.
100
Vo Act
80
60
40
20
20 40 60 0 ~ 8 0
FIGURE 3
Influence of the temperature on the activity of pigeon
(-) and hen (----) egg white lysozymes.
Activity values are plotted as percentage of those
obtained at 20". Studies have been performed in
potassium phosphate buffer, pH 3.3 and 0.14M ionic
strength, and pH 6.5 and 0.14M ionic strength. The
protein concentration in the incubation sample was
0.04 and 0.02 mg/mI for the pigeon and hen enzymes,
respectively. The heat treatment was performed for
10 min at different temperatures prior to the enzyme
assay.
data contrast with the acidic optimum pH
values observed for the lysozyme g (30).
Molecular weight
The molecular weight of the pigeon egg white
lysozyme has been determined by gel filtration
and SDS-polyacrylamide gel electrophoresis.
The obtained value from a Sephadex G-75
calibrated column is 13 500 Daltons. This value
has to be considered on the basis of the inter-
action of lysozyme with dextrans (12). Thus,
the molecular weight of the enzyme could be
slightly higher than the experimental value.
From the SDS-polyacrylamide gel electro-
phoresis it is possible to conclude the presence
of a single polypeptide chain for the pigeon
lysozyme with a molecular weight of 15000
24 2
Daltons. This value agrees with that reported
for different bird egg white lysozymes ( 2 8 , 3 1).
Absorbance properties
The U.V. spectrum of the pigeon enzyme shows
a maximum centered at 280nm and two
shoulders at 289 and 275nm. The AzaolA2m
absorbance ratio is 1.58. Studies performed
with the hen enzyme under the same conditions
reveals also a maximum at 280nm as well as
two shoulders at 289 and 274 nm with an AzsoI
Azm ratio of 1.54. The E:iAP6,,, based on dry
weight measurements is 1.5. This value is
coincident with that obtained from the amino
acid composition, according to the method of
Wetlaufer (32).
Circular dichroism studies
The CD-spectra of the pigeon egg white lyso-
zyme are given in Fig. 5 . In the far-u.v. region,
a minimum ellipticity value is observed at 208
nm. Secondary structure estimations according
to Chen et al. (19) and Greenfield & Fasman
(33) did not allow a fit of the experimental
curve with the theoretical values. This has been
attributed to a negative contribution in the
228-250nm region which would arise from the
tertiary structure of the protein. For this reason,
the computer fit has been restricted to the
range wavelength below 220 nm. Taking this
100 -
%Act -
80 -
60 -
40 -
2olJ
Effect of pH on the activity of pigeon (-) and
hen (- -- -) egg white Iysozyme. Activity values are
plotted as percentages of the maximum obtained.
Assays have been performed at 0.1 M ionic strength.
 Pigeon egg white lysozyme
into account the obtained secondary structure
composition, according to Chen et al. (19),
is 31%a-helix, 16% /3 structure, 53% aperiodic
Y -50 ordination and an average value of residues per
helical segment of 9. The calculated ellipticity
values from this composition closely fit the CD-
- 1 w - - 1 ~spectrum of the hen egg white lysozyme
-150 --?w (Fig. 5).
The CD-spectrum in the near-u.v. region
210 220 230 240 nm 280 270 280 290 3W (320-250 nm) shows a broad negative band
centered around 275nm as well as a shoulder
FIGURE 5 at 290nm.
CD spectra of pigeon 1-( and hen (----) egg
white Iysozyme in potassium phosphate buffer, pH Amino acid composition
6.5 and 0.14M ionic strength. Ellipticity values are The amino acid composition of lysozyme from
expressed in units of degree x cm' x dmol-' of
the pigeon egg white is given in Table 2. No free
residue. (-): These points correspond to the theoretical
values based on 31% &-helix, 16% 0-structure, 53%
-SH groups are available to 5,5'-dithiobis
aperiodic structure and 9 for the average value of (2 nitrobenzoic acid) in the presence of SDS.
residues per helical segment. (*) Scale for the ellipticity This result agrees also with that obtained for
values of the hen enzyme. other lysozymes (1 6,34).

NH2-terminal sequence
The NH,-terminal amino acid sequence of the
TABLE 2 pigeon egg white lysozyme is given in Fig. 6.
Amino acid composition of pigeon egg white lysozyme. The NH,-terminal residue of the protein is
Residues are the nearest integer value based on 130 lysine and the first 22 residues have been deter-
amino acids. Comparison with hen lysozyme c (16, 34) mined. At position 14, Phe was detected in low
and goose lysozyme g (44) yield but no other amino acid residue was
found.
Amino acid Pigeon Hen Goose

21 20
COOH-terminal sequence
Asx 17
Thr 6 7 12 Carboxypeptidase A digestion of the native
Ser 9 10 9 lysozyme released valine in a 95% yield after
GI x 10 5 15 15 h incubation time. Assays were performed
Pro 5 2 4 with about 6nmol protein. No other amino
G 1Y 11 12 20
Ala 8 12 15 5 10

8a 8 4 pigeon LYS-ASP-ILE-PRO-ARG-CYS-GLU-LEU-VAL-GLY-ILE-
CY s
Val 8 6 12 hen Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ale-Ale-Ale-

Met 1 2 3 duck I1 Tyr-Ser

Ile 6 6 13 chachalaca Ile-Tyr-Lys
Leu 6 8 7 15 20
TYr 4 3 9 pigeon -LEU-LYS-PHE- -GLY-PHE-ASX- -1YR-ARG-GLY-
Phe 3 3 3 hen -Met-Lys-Arg-His-Gly-Leu-Asp-Asn-Tyr-Arg-Gly-
His 2-3 1 5
duck I1 Leu
LYs 13 6 17
9-10 11 9 chachalece Tyr
'4%
TIP 3b 6 3 FIGURE 6
No. of residues 129-131 129 180 NH,-terminal sequence of pigeon lysozyme in com-
parison with those from hen (16, 34), duck I1 (2)
a Determined as cysteic acid after 24 h hydrolysis. and chachalaca (5). No amino acid derivative were
Determined spectrophotometrically. detected at positions 15 and 19.

243
J.G. Gavilanes et al.
acids were detected in significant yield. Carboxy- vertebrate lysozymes of known complete or
peptidase Y treatment of the performic acid partial sequence. At position 9 the pigeon has
oxidized lysozyme (6nmol) gives, after 1 h a conservative substitution (Ala to Val), which
incubation, 95% valine, 95% arginine, 60% is found in insect lysozymes. At positions 8, 12,
cysteic acid, 15% tyrosine and 10% leucine. The 17, 18 and 21 the pigeon enzyme has amino
proposed COOH-terminal sequence for the acids which have been seen in the lysozyrnes c
pigeon lysozyme is -Cys-Arg-Val. of other creatures. Thus, at position 8 pigeon
lysozyme is the same as in other birds, primates
and insects; at 12 as in mammals and insects;
at 17 as in insects; at 18 as in birds, tortoise and
DISCUSSION
primates; at 21 as in most birds and primates.
Two different types of lysozyme have been At positions 2, 3, 4, 10, 1 1 and 14 the pigeon
characterized in the egg white of birds. High has amino acids not seen in any other lyso-
levels of lysozyme c occur in the egg white of zyme. The changes at positions 3, 10 and 11
many species in the order Galliformes as well may be considered conservative relative to at
as in some species of the order Anseriformes least some other lysozymes (5,38-42).
(1, 35, 36). The second type, lysozyme g, has The carboxy-terminal sequence of all lyso-
been detected in the egg white of species from zymes c sequenced is -Cys-X-Leu or Val. This
nine different orders of birds (4). Egg white of is in agreement with the obtained result for the
species from 12 orders, including Columbi- pigeon lysozyme. The sequence for the pigeon
formes, did not react with antisera to either enzyme is -Cys-Arg-Val.
goose lysozyme g, chicken lysozyme c or duck Despite the observed sequential homology
lysozyme c in the Ouchterlony test (4). These between the hen and pigeon lysozymes, lack of
authors conclude that such egg white may reactivity of the pigeon enzyme with antiserum
contain lysozyme c and/or lysozyme g which to chicken lysozyme (4) has been described.
have evolved considerably from a common Nevertheless residues located at positions 5, 7
ancestor with the Galliformes or Anseriformes. and 13, involved at the antigenic site 1 of lyso-
Alternatively, another type of lysozyme may zymes according to Atassi & Lee (43), coincide
exist in their egg white. with the homologous positions of the hen
However, according to our results, the enzyme. These facts would indicate for Columbi-
pigeon egg white lysozyme may be considered formes an important degree of divergence with
type c. Certainly the thermal stability at acidic Galliformes and Anseriformes from a common
pH of the pigeon enzyme is significantly lower ancestor in spite of a common chicken type
than that observed for the hen enzyme. Never- lysozyme.
theless, the alkaline optimum pH value as well
as the molecular weight and electrophoretic ACKNOWLEDGEMENTS
mobility are coincident for both enzymes,
whereas lysozyme g shows an acidic optimum We wish to thank Dr A.M. Municio for his counsel
pH (30) and a higher molecular weight (27,37). in the course of this work as well as in the preparation
The specific activity of the pigeon enzyme and of this manuscript.
its content in the egg white agree also with the
proposed type c. The secondary structure of REFERENCES
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Address:
Dr. Jose G. Gavilanes
Departamento de Bioquimica
Facultad de Quimica
Universidad Complutense
Madrid-3
Spain
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