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Separation of insect hemolymph proteins by cascade-mode multiaffinity chromatography.

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Archives of Insect Biochemistry and Physiology 20:243-251 (1992)
Separation of Insect Hemolymph Proteins by
Cascade-Mode MuItiaff inity Chromatography
Preminda Samaraweera, JerkerPorath, and John H. Law
Centerfor Insect Science (P.S., J.H.L.) and Depurtment of Biochemistry (P.S., J.P., J.H.L.),
University of Arizona, Tucson
The hemolymph of the adult female Manduca sexta was fractionated by
cascade-mode multiaffinity chromatography (CASMAC) on a main-line tandem column chain containing Zn2+-TED, T-gel, Ni2+-DPA, and phenylsepharose and a side-line column containing Zn2+-DPA. The technique
separated some of the previously described major hemolymph proteins, and
yielded a number of fractions with simple composition. Some of these fractions contained only less abundant proteins of Manduca hemolymph. Thus,
it appears that CASMAC would be a very useful fractionation technique for
purification and characterization of the minor proteins of insect hemolymph.
@ 1992 Wiley-Llss, Inc.
Key wards: Manduca sexta, metal ion affinity, multiaffinity adsorption
Using conventional methods, it has been possible to isolate and characterize every major protein of the hemolymph of the adult hawkmoth, Manducu
sexta [l].There are, however, many additional proteins that can be seen in 2-D"
gels of this fluid. If we are to gain knowledge of the various minor proteins,
many of which may have vital functions, we must resort to new methodologies. While in theory it should be possible to isolate sufficient quantities of
the minor proteins from 2-D gels for N-terminal sequence, and perhaps for
production of antibodies, both of which should facilitate cloning and sequencing, in practice this is frequently difficult or impossible. The task would be
made easier if one could first fractionate hemolymph to yield less complex
Acknowled ments: This work was supported by the grant GM29238 from the National Institutes of He3th.
Received December 23,1991; accepted April 14,1992.
Address reprint requests to Dr. John H. Law, De artment of Biochemistry, Biological Sciences
West Building, Universityof Arizona, Tucson, d85721.
*Abbreviations used: CASMAC = cascade mode multiaffinity chromatography; 2-D = two
dimensional; D = Dalton; DPA = dipicolylamine agarose; IMA = immobilizedmetal ion affinity; IMAC = immobilized metal ion affinity chromatography; SDS = sodium dodecyl sulfate;
TED = tris-carboxymethylethylene diamine agarose; T-gel = thiophilic gel.
0 1992 Wiley-Liss, Inc.
Sarnaraweera et al.
mixtures, and then use simple gel electrophoresis for the final isolation. This
strategy also yields proteins in the native, rather than denatured, state.
Among the newer technologies that might be useful for this purpose are
IMAC [2]and the combination of IMAC with other affinity methods, for example, CASMAC [3]. IMAC has been demonstrated to be a useful separation
method for a variety of proteins [4-61, and CASMAC has been successfully
applied for the separation of components of human serum [7]. We recently
reported the use of a ferric ion IMAC method for a one-step isolation of insect
vitellins [8].
We will now report the use of CASMAC to separate proteins of adult hemolymph of Manduca sexta. The method yields several fractions of relatively simple composition, and some of the known major proteins have been identified
in these fractions. In addition, some previously undescribed proteins have
been revealed by this separation technique.
M . sextu eggs were supplied by Drs. J.P. Reinecke and J.S. Buckner, U.S.
Department of Agriculture, Fargo, ND. Animals were raised as previously
described [9].
Antibodies to M . sextutd Hemolymph Proteins
Rabbit polyclonal antibodies against apolipophorin I11 [ 101, microvitellogenin
[ll],transferrin [12], ommochrome-binding protein [13], and vitellogenin [14]
were prepared in this laboratory. Anti-serpin antibodies were kindly provided by
Dr. Michael Kanost of Department of Biochemistry, the University of Arizona.
Hemolymph Collection and Density Gradient Centrifugation
Adult M . sextu (40-50; male or female)were bled by the "flushing out" method
of Chino et al. [15] using 0.1 M sodium phosphate, 0.15 M NaCl, pH 7, that
contained 10 mM diisopropylfluorophosphate and 50 mM glutathione for injection into the moths. The hemolymph preparation was then subjected to KBr
density gradient centrifugation [16] at 50,000 rpm in a Beckman VTi 50 rotor
at 5°C for 16 h, and the lipoprotein-free subnatant was collected. The preparation was then dialyzed against 60 mM sodium phosphate, pH 7.5.
Chromatography Media
Phenyl-sepharose and butyl-sepharose were from Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). Preparation of DPA, TED, and T-gel have been
described [7,17,18]. DPA and TED, respectively, bound 45 and 58 pmol Cu2+
ions/ml adsorbent. Immobilization of metal ions (Ni2+or Zn2') was performed
by passing 50 ml of 20 mM metal ion solution through the column containing
DPA or TED (8-10 ml of gel). The column was then washed with distilled water
followed by starting buffer. Zn2+-DPA and Zn2+-TEDwere reloaded with
metal ions after each use because possible migration and leakage of the metal
ion in the bed cannot be followed by visual inspection. Ni2+-DPAwas used
without any reloading.
Hemolymph Proteins of Manduca sexfa
All chromatography runs were performed at room temperature in 1.5 cm x
10 cm columns (Bio-Rad Laboratories, Richmond, CA)at a flow rate of 1mb'min,
In order to determine the affinities of different proteins for different adsorbents preliminary experiments were conducted usin individual columns. The
bed volumes were as follows: Ni2+-DPA,8 ml; Zn'+-DPA, 8 ml; Zn2+-TED,
10 ml; butyl-sepharose, 14 ml; phenyl-sepharose, 14 ml; and T-gel, 15ml. Each
column was equilibrated with 50 mM sodium phosphate, pH 7.6, that contained 0.6 M K2S04 (buffer I). "Antichaotropic" sulfate was added to a high
concentration in order to suppress simple ionic interaction and to intensify
coordinate binding. Two milliliters of the lipophorin-free hemolymph prepared as above was brought into the same buffer by adding solid K2S04, dilution with water and pH adjustment, and applied to each column. The optical
absorbance at 280 nm of the hemolymph preparation was 5-6 absorbance
units. Therefore the total optical absorbance of the material used in all chromatographic runs amounted to 10-12 units. After washing the columns with
buffer I to remove unbound and loosely bound proteins, the columns containing Ni2+-DPA,Zn2+-DPA, and &?+-TED were developed with 50 mM
sodium phosphate, 0.6 M K2S04,0.1 M imidazole, pH 7.6 (buffer 11), followed
by 0.5.M sodium acetate, 0.5 M NaC1, pH 5.5 (buffer 111) (compared to sulfate,
acetate and chloride are located in the direction of increasing chaotropic behavior on the Hofmeister scale). Those containing butyl-sepharose, phenylsepharose, and T-gel were successively eluted with buffers of decreasing
polarity, namely, 50 mM sodium phosphate, pH 7.6 (buffer IV), 40% ethylene
glycol in buffer IV (buffer V), and 30% 2-propanol in buffer IV (buffer VI). The
elution of proteins from the columns was monitored by measuring the optical
absorbance at 280 nm. The proteins eluted on each development step were
analyzed by SDS-PAGE. On the basis of the preliminary investigations columns containing Zn2+-TED, T-gel, Ni2+-DPA, and phenyl-sepharose were
connected in series to give a main-line tandem column (Fig. 1). The tandem
column equilibrated in buffer I was loaded with the hemolymph preparation
(brought into buffer I as above) and unbound and loosely bound proteins were
washed out. This initial washing was routinely carried out with 180 ml of buffer
I; all the other chromatography steps used 60 ml of the respective buffers. In
all cases, 3 ml fractions were collected. The non-interacting fraction was denoted
fraction A. Individual columns were then disconnected and developed separately. The column containing Zn2+-TEDwas successively developed with
buffers I1 and I11 to give fractions B and C, respectively. T-gel was washed with
buffer IV and the effluent was directed to a column containing Zn2+-DPA.
The fraction which was eluted without any interaction with Zn2+-DPAwas
denoted D. The two columns were then disconnected, and T-gel was eluted
with buffer V followed by buffer VI to give fractions E and F. Zn2'-DPA was
washed with buffers I1 and 111. These two fractions were denoted G and H,
res ectively. Ni2+-DPAwas developed in the same way as Zn2'-TED and
Zn -DPA, i.e., with buffers I1 and 111, to give fractions I and J. Phenylsepharose was successively eluted with buffers IV, V, and VI to obtain fractions K, L, and M.
Samaraweera et ill.
Fig. 1. Schematic drawing of the arrangement of the tandem columns. The main-line consisting of Zn+’-TED, 1-Gel, Ni’+-DPA, and phenyl-sepharose (Phe.Seph.) is shown on the left
side of the diagram. 4 indicates elution of columns, and 3 indicates disconnection of the
bed. The Roman numerals I-VI represent buffer systems used to develop the columns, and
A-M indicate the fractions obtained. For example, the 1- el column was disconnected from
the tandem column, and eluted with buffer IV into the Zn’+-DPA column. The material which
did not bind to either column was designated fraction D. The 1-gel column was again disconnected, and eluted with buffers V and VI to give fractions E and F, respectively.
SDS-Polyacrylamide Gel Electrophoresis
SDS-polyacrylarnide slab gel electrophoresis was carried out as described
by Laemmli 1191. The proteins were routinely analyzed in 4-15% gradient gels
under reducing conditions.
The proteins on SDS-polyacrylamide gels were slectrophoretically blotted
onto nitrocellulose membranes [ZO]in a Hoefer Scientific Instruments TE-52
Hernolymph Proteinsof Manduca sexta
transphor electrophoresis unit (San Francisco, CA). Immunoblotting was per
formed using rabbit anti-protein antibodies and Vectastain ABC reagent
kit (Vector Laboratories Inc. Burlingame, CA) as recommended by the manufacturer.
Affinities of Munducu Hernolymph Proteins for Different Adsorbents
The initial studies using individual columns were performed to determine
adsorption behavior of hemolymph proteins toward the different adsorbents.
Both quantitative and qualitative differences in adsorptions were observed
between different gels. Under the experimental conditions used, phenylsepharose and Ni2+-DPAbound more than 80% of applied hernolymph proteins, whereas T-gel, butyl-sepharose, and %'+-TED adsorbed some 20-30%,
Zn2+-DPAadsorbed about 60% of the hernolymph proteins.
Adsorption of Hernolymph Proteins by Tandem Column
The different adsorbents in the tandem column (Fig. I) were arranged
depending on their affinities for hemolymph proteins. Since Zn2+-TED
adsorbed the least amount of protein this gel was placed first in the tandem
column. The T-gel adsorbed some additional proteins, and therebre, was placed
next to Zn2"-TED column. The Ni2+-DPAcolumn was inserted next and
phenyl-sepharose which adsorbed highest amount of protein was placed Iast.
Figure 2 shows a typical composite elution profile of the chromatography of
adult female Munducu hemolymph as described in the Materials and Methods
section. The amounts of proteins recovered in the fractions (as the percentage
of applied proteins) are summarized in Table 1. The majority of the proteins
was bound to Ni2+-DPAand eluted in fractions I and J. Zn'+-TED bound
(fractionsB and C) only 10%of the applied proteins. This value contrasts with
the finding using the individual column, where it bound about 20% of the
applied proteins. Although it is unlikely, the reduction of binding of proteins
by Zn2+-TED
in the tandem column may be due to extended washing involved
in the procedure. The results of SDS-PAGE of eluted fractions are shown in
Figure 3. The intensities of the protein bands in Figure 3 are not indicative of
the proportion of the proteins in the original hemolymph preparation: in order
to make the bands visible in the Coomassie blue stained gel, certain fractions
(E and F, for example) required loading of as much as 60-70% of the preparation, while some (fractions I and J) yielded sufficiently intense bands with
less than 5% of the fraction*Some of the protein bands present in SDSpolyacrylamide gel were identified by immunoblotting, and are indicated in
the same figure. Only a few proteins were present in fraction A, and microvitellogenin was the major constituent. Fraction B contains a number of proteins,
but, as can be seen when compared to the applied hemolymph preparation
(Fig. 3, lane He), almost all of them seem to be minor constituents. Similarly,
fractions D-H contained mainly minor hemolymph proteins. Transfenin and
vitellogenin are the major proteins in fraction I while traces of insecticyanin
are also present. More than 90% of insecticyanin together with some vitellogenin
and ommochrome-binding protein [13]were eluted in fraction J. Serpin is a
Sarnaraweera et al.
4 0
Fig. 2. The composite elution profile obtained on fractionation of adult female Manduca
hemolymph by CASMAC. Arrows with I-VI indicate starting elution positions with respective
buffers. A-M represent peaks corresponding to each fraction.
constituent in fraction K, and apolipophorin I11 was eluted in fraction L in
a relatively pure form. While vitellogenin was observed in many fractions the
amounts present in fractions E, G, and H represent only a small percentage.
The majority of the protein is present in fractions I and J. The distribution
of vitellogenin in different fractions may be due to its heterogeneity in terms
of phosphorylation and glycosylation. An unidentified protein with approximate M, 30 kD is eluted in fraction M in a considerably purified form. Again,
this protein seems to represent a less abundant constituent in Munducu hemolymph. The results of the fractionation of male hemolymph are very similar
to that of female hemolymph except that peak J is higher than peak I in the
chromatogram, and vitellogenin subunits and microvitellogenin bands are absent in SDS-polyacrylamide gel for male hemolymph (not shown).
TABLE 1. Recoveries of Proteins in the Fractions Obtained From CASMAC (Expressedas the
% of applied sample)
% recovery
Hernolymph Proteins of Manduca sexfa
Fig. 3. SDS-PAGE of the fractions collected on CASMAC. The analysiswas performed on 4-15%
gradient gels under reducingconditions. Lanes: Mo, molecularweight markers; He, hernolymph
subnatant after density gradient centrifugation; A-M, materials in fractions A-M, respectively.
Proteins identified by irnmunoblotting are indicated with arrows. a, vitellogenin large subunit;
b, transferrin; c, vitellogenin small subunit; d, rnicrovitellogenin; e, insecticyanin; f , apolipophorin I l l ; g, ornrnochrorne-bindingprotein; h, serpin.
Although a considerable body of knowledge has been accumulated about
Munducu hemolymph proteins [l],most of the past studies were focused on
the major components. The fact that the less abundant proteins have drawn
little attention can be mainly attributed to the difficulty of purification which
is a prerequisite for their characterization and understanding of their biological role. The present investigation was undertaken to explore the possibility
that CASMAC will yield less complex mixtures of hemolymph proteins and
facilitate purification of minor proteins. To achieve this objective we utilized
IMA gels combined with thiophilic and hydrophobic gels in a manner described
by Porath and Hansen [7]. Since we lack an understanding of the interaction
of M. sextu hemolymph proteins with different affinity adsorbents, the choice
of the adsorbents and the order in which they were arranged in the tandem
column were empirical. With the exception of vitellogenin, we were able to
separate many of the known adult Munducu hemolymph proteins from each
other. For example, the procedure resolved microvitellogenin, transferrin,
insecticyanin, and apolipophorin I11 into different fractions (fractions A, I, J,
and L, respectively). The distribution of vitellogenin in more than one fraction may reflect heterogeneity in its composition. We have previously reported
a simple one-step IMAC purification of vitellogenin [8].The recovery of the
Samaraweera et id.
CASMAC was high; we recovered more than 90% of the proteins (in terms of
absorbance at 280 nm) applied to the tandem column chain. This less than
100% recovery may be due to insertion of T-gel and phenyl-sepharose in the
tandem column; during the initial tests using individual adsorbents the IMA
gels yielded close to 100% recovery while with T-gel and phenyl-sepharose
only 85-90% of the proteins could be recovered. We were also able to separate
by CASMAC some less abundant proteins into simple mixtures. Repetition of
the experiment a number of times yielded the same results. Currently we are
attempting to purify minor proteins from these fractions.
Immobilized metal ion affinity gels are prepared by coupling a ligand to an
insoluble matrix, and coordinating metal ions to the ligand. Although IMAC
is a rather new technique it may have wide applications in separation of a
variety of biomolecules because one can combine a large number of ligands
and a large number of metal ions to obtain an enormous number of affinity
adsorbents. Variations in binding and elution of proteins are possible by changing the experimental conditions (ionic strength, pH, etc.). Here we used one
hydrophobic adsorbent, one thiophilic adsorbent, and three kinds of IMA gels.
Further resolution of hemolymph proteins should be possible by inserting more
IMA gels and other types of adsorbents (ion exchange gels, for example) into
the main-line tandem or as side-line columns. The separation can be further
improved by introducing salt or pH gradients to displace bound proteins.
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proteins. Adv Insect Physiol22,299 (1990).
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S193 (1987).
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cascaded, separating, mode, protein, multiaffinity, chromatography, insect, hemolymph
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