Vol. 74, No. 4, 1977
BIOCHEMICAL
AND BiOPHYSICAL
RESEARCH COMMUNICATIONS
LYSOZYME-AGAROSE INTERACTION J.M.Ferndndez-Sousa Department
of Biochemistry.
Complutensis Received
November
and R-Rodriguez Faculty
University.
Madrid.
of Sciences Spain
16,1976
Summary. It has been observed that the elution volumes of hen eggwhite lysozyme on agarose columns exceeded by far the total volumes of the corresponding columns. Similar results have been obtained also on polyacrylamide-agarose gel columns. This anomalous behaviour of lysozyme on agarose columns is interpreted in terms of an interaction between the enzyme and agarose which resembles in some way the physiological substrates of lysozyme. It has been already reported pancreatic a-amylase elutes later volumes on gel filtration The possible retention was ruled the ionic ed to
that than
chromatography of these proteins
lysozyme and before the total
in dextran columns (l-4). by an ion exchange mechanism
out as the elution volumes were found to be independent of strength. Thus, this anomalous behaviour has been attribut-
an interaction While modified
of lysozyme and a-amylase with dextran gels (3). dextrans used as sieves are three-dimensional agarose is a linear chain polymer of -Dchains,
networks of glucan galactose6(1+4)-3,6-anhydro-L-galactoseu(l+3)-. the structure of bacterial cell walls chains built muramic acid similar tention
hen egg-white expected but
up of alternating residues linked
is
On the other hand, composed of polysaccharide
N-acetylglucosamine and N-acetyl throughout B(1+4) linkages, and thus
to agarose. Accordingly, if the reason of hen egg lysozyme on dextran gels is
for the
the abnormal interaction
re -
between the enzyme and the polysaccharide structure, a stronger interaction would be expected with agarose and consequently greater elution volumes of lysozyme on agarose filtration chromatography than
on dextran gel columns. These considerations led us to investigate lysozyme with agarose through the comparative enzyme in gel
filtration
chromatography
MATERIALS AND METHODS Hen egg-white lysozyme, cell walls from Michococcun
Copyright 0 1977 by Academic All rights of reproduction in any
Press, Inc. form reserved.
horse
heart
.fLqbodeikXicub
1426
the interaction behaviour of the
on different
of
media.
cytochrome c, bacterial and 2-mercaptoethanol
were
ISSN
0006-291X
BIOCHEMICAL
Vol. 74, No. 4, 1977
obtained
from Sigma Chemical
and Blue Dextran Biogel A, 0.5m,
(St.Louis,USA).
Sephadex G-75,fine,
were purchased from Pharmacia (10% agarose) was from Bio-Rad
Co.
(Upsala, Sweden). Lab. (Richmond,USA).
Ultrogel AcA 54 (5% acrylamide (Sweden). All other chemicals 6.3,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and 4% agarose) was from LKB Products were reagent grade.
Sephadex was suspended in 0.05 M potassium phosphate and allowed to swell for 2-3 days at room temperature.
swollen Ultrogel
buffer, The
pH
Sephadex suspension as well as the comercial suspensions of AcA 54 and Biogel A were degassed before poured into the
(lx50cm) columns, for chromatography. were calculated
and then equilibrated Void (V,) and total
with
Blue
Dextran
with (Vt)
the same solutions used volumes of the columns
and ammonium hydroxide
respectively.
A solution of 2.2 mg of hen egg-white lysozyme and 2.2 mg of horse heart cytochrome c in 250 ul was applied to the Sephadex G-75, Biogel A and Ultrogel AcA 54 columns, previously equilibrated, and eluted with a) 0.05 M potassium phosphate buffer, pH 6.3,(I'/2=0.07); b) 0.05 M phosphate buffer, pH 6.3, containing 0.28 M KCl(T/2=0.35); c) 0.05 M phosphate d) 0.05 M phosphate phosphate ethanol.
buffer,
buffer, buffer, pH 6.3,
pH 6.3, pH 6.3,
containing containing
containing
1.0 M KCl(I'/2=1.07) 6 M urea and e)0.05
6 M urea
Lysozyme was determined either absorbance at 280 nm or by its lytic
and 25 mM mercapto
; M -
spectrophotometrically by its activity towards Michococcun
cell walls at pH 6.3 and 0.1 ionic strength. Cyto chrome c was determined by its absorbance at either 280 nm or 410 nm
Cynodeihticud
or 550 nm, according
to the
experiment.
Discrimination between cytochrome c and lysozyme was occasionally done on polyacrylamide gel electrophoresis performed according to Panyim and Chalkley (5), using
Amido Black
dye for
gel
staining.
RESULTS AND DISCUSSION Fig.la heart
shows a typical
cytochrome
elution
pattern
c and hen egg-white
lysozyme
of a mixture
of horse
from a Sephadex
G-75
column: this elution profile shows clearly that both proteins overlap partially in spite of the differences in their molecular weight. It is interesting to note, however, that hen lysozyme (mw.14307) elutes slightly later than horse cytochrome c (mw.12398). Concerning the mechanisms underlying this fact, it has previously reported that basic proteins such as horse heart cytochrome c (p1 10.8) (6) and hen lysozyme
(p1 11-O-11.3)
(7) eluted
later
1427
than
expected
from dextran
Vol. 74, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.6-
SEPHADEX
G-75
ULTROGEL
AcA
54
-5
BIOGEL
A 0.5m -10
-5
25
30
35
40
45
50
55
60
65
rnl
Figure 1. Elution profiles of cytochrome c and lysozyme from Sephadex G-75 (a), Ultrogel AcA 54 (b) and Biogel A (c). Total recorded proteins are given by the continuous line. Cytochrome c was evaluated by absorption at 550 nm ($@:l') and lysozyme was determined by its lytic activity (m).
gels due to weak ion-exchange properties of the gels (1). This result has been later attributed to a specific interaction of lysozyme with the dextran gel matrix rather than to the more basic character of the molecule of lysozyme (3,4). Elution profiles of the mixture of horse cytochrome c and hen lysozyme from columns of Ultrogel AcA 54 (polyacrylamide/agarose) and Biogel A 0.5m (agarose) (figs.lb and lc, respectively)clearly show the high resolution of both proteins. Cytochrome c elutes from these columns at the expected volume, but hen lysozyme elutes with a volume
1428
Vol. 74, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C”3
co kn
CH3
$0
Cl+ll
...+iCVH ,,“,:.+o+J CH co no-
_ A0
un-
CELL
b43
. . ..&.e.
=
/-
WALL
NCI
xao..
co
POLYSACCHARIDE
=“3
“07---.JJo~o... qon
NW
Nbl
CO &I3
CO dH3
CHITIN
AGAROSE
Figure 2. Fragments of the structure of bacterial saccharide, chitin and agarose matrix.
cell
wall
poly
-
greater than the total volume of the corresponding agarose columns. Moreover, lysozyme elutes from Biogel A (10% agarose) columns with a greater volume than from the Ultrogel AcA 54 (5% polyacrylamide and 4% agarose) columns. Ionic strength (0.07) and pH (6.3) of the buffer used in these gel filtration chromatography experiments are those at which could
lysozyme exhibits maximal be explained on the basis
activity (8). of a specific
Thus, these interaction
results of lyso
-
zyme with the agarose gel matrix, possibly due to the similarity of the structure among agarose and bacterial cell wall polysaccharides and chitin. In fig.2 a fragment of these structures is given. If the mixture of horse cytochrome c and hen lysozyme is chromate graphed on agarose columns equilibrated with 0.05 M potassium phos phate buffer, pH 6.3, containing 6 M urea, lysozyme is eluted slightly later than cytochrome c and before the total volume of the corresponding columns (Table 1). This fact could be explained through a weak interaction of lysozyme in 6 M urea with the agarose matrices that account
for
an elution
volume
higher
than
that
is in coincidence with a remanent enzyme activity tributed to the remaining unfold of the protein 9-10 M urea
(9,lO). 1429
of cytochrome
c.
It
of about 3-5% atin the presence of
Vol. 74, No. 4, 1977
Elution
Elution Solvent
BIOCHEMICAL
volumes lysozyme
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 of horse cytochrome c and hen egg-white on gel chromatography columns Sephadex G-75 Ultrogel AcA 54 Biogel A, 0.5 m Vt=34.6 ml Vt=37.5 ml Vt=3B.0 ml
Protein
a
Cytochrome Lysozyme
c
b
Cytochrome
c
Lysozyme
28.2 ml 30.9
36.8 ml
37.5
46.4
57.2
27.6 29.7
35.7 41.4
35.5 41.7
C
Cytochrome Lysozyme
c
27.5 29.5
35.5 41.2
35.5 41.4
d
Cytochrome Lysozyme
c
27.8 28.8
34.6 36.6
31.5 34.7
Cytochrome
c
27.6
34.5
32.1
28.2
35.4
33.6
e
Lysozyme
Similarly,
it
has been reported
that
the activity
ml
of hen lysozyme
at 0.35 ionic strength decreased to S-10% of the maximal activity at 0.07 ionic strenth, pH 6.25. Thus, Davies et al. (8)suggest that the electrostatic interaction of lysozyme with the bacterial cell wall polysaccharide used as substrate decreased as the ionic strength of the medium raised. Effectively, the elution patterns of the mixture cytochrome could also lysozyme table 1 variation therefore, strengths. columns retention matrices If carried
c-lysozyme from agarose columne (see fig.1 and table 1) be interpreted in terms of the remanent activity of hen
at 0.35 ionic strength. However, it can also be observed in that by increasing the ionic strength up to 1.1, a very low of the elution volume is registered and it can be concluded, that lysozyme exhibits some activity even at high ionic On the other hand, this retention of lysozyme on agarose at high ionic strengths also rule out the possibility that the is due to ion exchange properties of the polysaccharide of the gels. the gel chromatography of the mixture cytochrome c-lysozyme is out in the presence of 6 M urea containing 25 mM 2-mercapto -
ethanol , a different elution profile presence of 25 mM 2-mercaptoethanol,
1430
is obtained (Table 1). In the disulfide bridges of the enzyme
BIOCHEMICAL
Vol. 74, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are reduced (7) and lysozyme is totally devoid of lytic activity. Thus, chromatography on agarose columns with 2-mercaptoethanol shows that lysozyme regains its overlap with cytochrome c. All these facts suggest that the anomalous behaviour of native lysozyme interaction saccharide structure
on agarose
gels
can be explained
on the basis
of a specific
between the active center of the enzyme and the poly matrix due to a certain resemblance of the agarose with those of the bacterial cell wall polysaccharides and
chitin. Concerning
the
functional
groups
involved
in these
interactions,
acetamide and 8(1+4) linkage are the main groups offering significant challenges. Both groups are necessary for the lytic activity of lysonevertheless, amide groups have no equivalent in the agarose zyme (7); structure
whereas
8(1+4)linkages
are common to the
lysozyme
and to the agarose matrix simmilarities in structure
(fig.2). This fact, and conformation,
that with
with lysozyme due to their cell wall polysaccharides.
agarose gels interact chitin and bacterial Chromatography
on agarose
selective method to purify ing any contamination with the gel matrix; molecules that
substrates
jointly with other allows one to consider resemblance
columns has been described
to be a
lysozymes due to these facts, thus other proteins that do not interact
avoid with
-
simultaneously, lysozymes are obtained free of small should elute at approximately the Vt of the column(l1).
REFERENCES 1. Miranda,F., 2. Gelotte,B. ion in gel 3. Whitaker,J.R., 4. Kramer,K.J.,
H.Rochat
and S.Lissitsky,
Quoted by P.Flodin, filtration", Upsala Anal.Chem., Ph.D.Thesis,
J.Chromatog.,
1,
142 (1962)
in "Dextran gels and their (Sweden), Pharmacia, 1962.
35, 1950 (1963) University of Arizona
applicat-
(1971)
5. Panyin,S. and R.Chalkley, Arch.Biochem.Biophys., 130, 337 (1969) 6. Lemberg,P. and Barret,J., In "Cytochromes" pp.122-216, Academic Press (New York), 1973. 7. Imoto,T., L.N.Johnson, A.C.T.North, D.C.Phillips and J.A.Rupley, In "The Enzymes", ~01.7, pp.665-868 (ed.P.D.Boyer), Academic Press, New York, 1972 A.Neuberger and B.M.Wilson, Biochim.Biophys.Acta, 178, 8. Davies,R.C., 293-305 (1969) 9. Hamaguchi,K. and A.Kurono, J.Biochem.(Tokyo) 54, 111 (1963) Biochim.Biophys.Acta, 178, 293 (1969) lO.Steiner,R.F., ll.Rodriguez,R. Ph.D.Thesis. Complutensis University, Madrid (1976)
1431
BIOCHEMICAL
AND BiOPHYSICAL
RESEARCH COMMUNICATIONS
LYSOZYME-AGAROSE INTERACTION J.M.Ferndndez-Sousa Department
of Biochemistry.
Complutensis Received
November
and R-Rodriguez Faculty
University.
Madrid.
of Sciences Spain
16,1976
Summary. It has been observed that the elution volumes of hen eggwhite lysozyme on agarose columns exceeded by far the total volumes of the corresponding columns. Similar results have been obtained also on polyacrylamide-agarose gel columns. This anomalous behaviour of lysozyme on agarose columns is interpreted in terms of an interaction between the enzyme and agarose which resembles in some way the physiological substrates of lysozyme. It has been already reported pancreatic a-amylase elutes later volumes on gel filtration The possible retention was ruled the ionic ed to
that than
chromatography of these proteins
lysozyme and before the total
in dextran columns (l-4). by an ion exchange mechanism
out as the elution volumes were found to be independent of strength. Thus, this anomalous behaviour has been attribut-
an interaction While modified
of lysozyme and a-amylase with dextran gels (3). dextrans used as sieves are three-dimensional agarose is a linear chain polymer of -Dchains,
networks of glucan galactose6(1+4)-3,6-anhydro-L-galactoseu(l+3)-. the structure of bacterial cell walls chains built muramic acid similar tention
hen egg-white expected but
up of alternating residues linked
is
On the other hand, composed of polysaccharide
N-acetylglucosamine and N-acetyl throughout B(1+4) linkages, and thus
to agarose. Accordingly, if the reason of hen egg lysozyme on dextran gels is
for the
the abnormal interaction
re -
between the enzyme and the polysaccharide structure, a stronger interaction would be expected with agarose and consequently greater elution volumes of lysozyme on agarose filtration chromatography than
on dextran gel columns. These considerations led us to investigate lysozyme with agarose through the comparative enzyme in gel
filtration
chromatography
MATERIALS AND METHODS Hen egg-white lysozyme, cell walls from Michococcun
Copyright 0 1977 by Academic All rights of reproduction in any
Press, Inc. form reserved.
horse
heart
.fLqbodeikXicub
1426
the interaction behaviour of the
on different
of
media.
cytochrome c, bacterial and 2-mercaptoethanol
were
ISSN
0006-291X
BIOCHEMICAL
Vol. 74, No. 4, 1977
obtained
from Sigma Chemical
and Blue Dextran Biogel A, 0.5m,
(St.Louis,USA).
Sephadex G-75,fine,
were purchased from Pharmacia (10% agarose) was from Bio-Rad
Co.
(Upsala, Sweden). Lab. (Richmond,USA).
Ultrogel AcA 54 (5% acrylamide (Sweden). All other chemicals 6.3,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and 4% agarose) was from LKB Products were reagent grade.
Sephadex was suspended in 0.05 M potassium phosphate and allowed to swell for 2-3 days at room temperature.
swollen Ultrogel
buffer, The
pH
Sephadex suspension as well as the comercial suspensions of AcA 54 and Biogel A were degassed before poured into the
(lx50cm) columns, for chromatography. were calculated
and then equilibrated Void (V,) and total
with
Blue
Dextran
with (Vt)
the same solutions used volumes of the columns
and ammonium hydroxide
respectively.
A solution of 2.2 mg of hen egg-white lysozyme and 2.2 mg of horse heart cytochrome c in 250 ul was applied to the Sephadex G-75, Biogel A and Ultrogel AcA 54 columns, previously equilibrated, and eluted with a) 0.05 M potassium phosphate buffer, pH 6.3,(I'/2=0.07); b) 0.05 M phosphate buffer, pH 6.3, containing 0.28 M KCl(T/2=0.35); c) 0.05 M phosphate d) 0.05 M phosphate phosphate ethanol.
buffer,
buffer, buffer, pH 6.3,
pH 6.3, pH 6.3,
containing containing
containing
1.0 M KCl(I'/2=1.07) 6 M urea and e)0.05
6 M urea
Lysozyme was determined either absorbance at 280 nm or by its lytic
and 25 mM mercapto
; M -
spectrophotometrically by its activity towards Michococcun
cell walls at pH 6.3 and 0.1 ionic strength. Cyto chrome c was determined by its absorbance at either 280 nm or 410 nm
Cynodeihticud
or 550 nm, according
to the
experiment.
Discrimination between cytochrome c and lysozyme was occasionally done on polyacrylamide gel electrophoresis performed according to Panyim and Chalkley (5), using
Amido Black
dye for
gel
staining.
RESULTS AND DISCUSSION Fig.la heart
shows a typical
cytochrome
elution
pattern
c and hen egg-white
lysozyme
of a mixture
of horse
from a Sephadex
G-75
column: this elution profile shows clearly that both proteins overlap partially in spite of the differences in their molecular weight. It is interesting to note, however, that hen lysozyme (mw.14307) elutes slightly later than horse cytochrome c (mw.12398). Concerning the mechanisms underlying this fact, it has previously reported that basic proteins such as horse heart cytochrome c (p1 10.8) (6) and hen lysozyme
(p1 11-O-11.3)
(7) eluted
later
1427
than
expected
from dextran
Vol. 74, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1.6-
SEPHADEX
G-75
ULTROGEL
AcA
54
-5
BIOGEL
A 0.5m -10
-5
25
30
35
40
45
50
55
60
65
rnl
Figure 1. Elution profiles of cytochrome c and lysozyme from Sephadex G-75 (a), Ultrogel AcA 54 (b) and Biogel A (c). Total recorded proteins are given by the continuous line. Cytochrome c was evaluated by absorption at 550 nm ($@:l') and lysozyme was determined by its lytic activity (m).
gels due to weak ion-exchange properties of the gels (1). This result has been later attributed to a specific interaction of lysozyme with the dextran gel matrix rather than to the more basic character of the molecule of lysozyme (3,4). Elution profiles of the mixture of horse cytochrome c and hen lysozyme from columns of Ultrogel AcA 54 (polyacrylamide/agarose) and Biogel A 0.5m (agarose) (figs.lb and lc, respectively)clearly show the high resolution of both proteins. Cytochrome c elutes from these columns at the expected volume, but hen lysozyme elutes with a volume
1428
Vol. 74, No. 4, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
C”3
co kn
CH3
$0
Cl+ll
...+iCVH ,,“,:.+o+J CH co no-
_ A0
un-
CELL
b43
. . ..&.e.
=
/-
WALL
NCI
xao..
co
POLYSACCHARIDE
=“3
“07---.JJo~o... qon
NW
Nbl
CO &I3
CO dH3
CHITIN
AGAROSE
Figure 2. Fragments of the structure of bacterial saccharide, chitin and agarose matrix.
cell
wall
poly
-
greater than the total volume of the corresponding agarose columns. Moreover, lysozyme elutes from Biogel A (10% agarose) columns with a greater volume than from the Ultrogel AcA 54 (5% polyacrylamide and 4% agarose) columns. Ionic strength (0.07) and pH (6.3) of the buffer used in these gel filtration chromatography experiments are those at which could
lysozyme exhibits maximal be explained on the basis
activity (8). of a specific
Thus, these interaction
results of lyso
-
zyme with the agarose gel matrix, possibly due to the similarity of the structure among agarose and bacterial cell wall polysaccharides and chitin. In fig.2 a fragment of these structures is given. If the mixture of horse cytochrome c and hen lysozyme is chromate graphed on agarose columns equilibrated with 0.05 M potassium phos phate buffer, pH 6.3, containing 6 M urea, lysozyme is eluted slightly later than cytochrome c and before the total volume of the corresponding columns (Table 1). This fact could be explained through a weak interaction of lysozyme in 6 M urea with the agarose matrices that account
for
an elution
volume
higher
than
that
is in coincidence with a remanent enzyme activity tributed to the remaining unfold of the protein 9-10 M urea
(9,lO). 1429
of cytochrome
c.
It
of about 3-5% atin the presence of
Vol. 74, No. 4, 1977
Elution
Elution Solvent
BIOCHEMICAL
volumes lysozyme
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1 of horse cytochrome c and hen egg-white on gel chromatography columns Sephadex G-75 Ultrogel AcA 54 Biogel A, 0.5 m Vt=34.6 ml Vt=37.5 ml Vt=3B.0 ml
Protein
a
Cytochrome Lysozyme
c
b
Cytochrome
c
Lysozyme
28.2 ml 30.9
36.8 ml
37.5
46.4
57.2
27.6 29.7
35.7 41.4
35.5 41.7
C
Cytochrome Lysozyme
c
27.5 29.5
35.5 41.2
35.5 41.4
d
Cytochrome Lysozyme
c
27.8 28.8
34.6 36.6
31.5 34.7
Cytochrome
c
27.6
34.5
32.1
28.2
35.4
33.6
e
Lysozyme
Similarly,
it
has been reported
that
the activity
ml
of hen lysozyme
at 0.35 ionic strength decreased to S-10% of the maximal activity at 0.07 ionic strenth, pH 6.25. Thus, Davies et al. (8)suggest that the electrostatic interaction of lysozyme with the bacterial cell wall polysaccharide used as substrate decreased as the ionic strength of the medium raised. Effectively, the elution patterns of the mixture cytochrome could also lysozyme table 1 variation therefore, strengths. columns retention matrices If carried
c-lysozyme from agarose columne (see fig.1 and table 1) be interpreted in terms of the remanent activity of hen
at 0.35 ionic strength. However, it can also be observed in that by increasing the ionic strength up to 1.1, a very low of the elution volume is registered and it can be concluded, that lysozyme exhibits some activity even at high ionic On the other hand, this retention of lysozyme on agarose at high ionic strengths also rule out the possibility that the is due to ion exchange properties of the polysaccharide of the gels. the gel chromatography of the mixture cytochrome c-lysozyme is out in the presence of 6 M urea containing 25 mM 2-mercapto -
ethanol , a different elution profile presence of 25 mM 2-mercaptoethanol,
1430
is obtained (Table 1). In the disulfide bridges of the enzyme
BIOCHEMICAL
Vol. 74, No. 4, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
are reduced (7) and lysozyme is totally devoid of lytic activity. Thus, chromatography on agarose columns with 2-mercaptoethanol shows that lysozyme regains its overlap with cytochrome c. All these facts suggest that the anomalous behaviour of native lysozyme interaction saccharide structure
on agarose
gels
can be explained
on the basis
of a specific
between the active center of the enzyme and the poly matrix due to a certain resemblance of the agarose with those of the bacterial cell wall polysaccharides and
chitin. Concerning
the
functional
groups
involved
in these
interactions,
acetamide and 8(1+4) linkage are the main groups offering significant challenges. Both groups are necessary for the lytic activity of lysonevertheless, amide groups have no equivalent in the agarose zyme (7); structure
whereas
8(1+4)linkages
are common to the
lysozyme
and to the agarose matrix simmilarities in structure
(fig.2). This fact, and conformation,
that with
with lysozyme due to their cell wall polysaccharides.
agarose gels interact chitin and bacterial Chromatography
on agarose
selective method to purify ing any contamination with the gel matrix; molecules that
substrates
jointly with other allows one to consider resemblance
columns has been described
to be a
lysozymes due to these facts, thus other proteins that do not interact
avoid with
-
simultaneously, lysozymes are obtained free of small should elute at approximately the Vt of the column(l1).
REFERENCES 1. Miranda,F., 2. Gelotte,B. ion in gel 3. Whitaker,J.R., 4. Kramer,K.J.,
H.Rochat
and S.Lissitsky,
Quoted by P.Flodin, filtration", Upsala Anal.Chem., Ph.D.Thesis,
J.Chromatog.,
1,
142 (1962)
in "Dextran gels and their (Sweden), Pharmacia, 1962.
35, 1950 (1963) University of Arizona
applicat-
(1971)
5. Panyin,S. and R.Chalkley, Arch.Biochem.Biophys., 130, 337 (1969) 6. Lemberg,P. and Barret,J., In "Cytochromes" pp.122-216, Academic Press (New York), 1973. 7. Imoto,T., L.N.Johnson, A.C.T.North, D.C.Phillips and J.A.Rupley, In "The Enzymes", ~01.7, pp.665-868 (ed.P.D.Boyer), Academic Press, New York, 1972 A.Neuberger and B.M.Wilson, Biochim.Biophys.Acta, 178, 8. Davies,R.C., 293-305 (1969) 9. Hamaguchi,K. and A.Kurono, J.Biochem.(Tokyo) 54, 111 (1963) Biochim.Biophys.Acta, 178, 293 (1969) lO.Steiner,R.F., ll.Rodriguez,R. Ph.D.Thesis. Complutensis University, Madrid (1976)
1431
