From the
A heme-binding protein has been isolated and characterized from
both the hemolymph and oocytes of the blood-sucking insect,
Rhodnius prolixus. The protein from both sources is identical
in most aspects studied. The Rhodnius heme-binding protein
(RHBP) is composed of a single 15-kDa polypeptide chain coiled in a
highly
RHBP from
hemolymph is not saturated with heme and promptly binds heme added to
the solution. The oocyte protein, on the other hand, is fully saturated
and is not capable of binding additional heme.
Hematophagy has evolved independently in several insect orders
and a great diversity of ways to digest and use blood have arisen
during the course of evolution
(1) . In general, blood feeding
is characterized by ingestion of enormous amounts of blood in a single
meal, usually comprising several times the animal's own weight
(2) .
A special problem generated by having vertebrate blood
as the sole food source is the large amount of free hemin that is
produced upon digestion of hemoglobin. Hemin is known to stimulate
lipid peroxidation
(3) as a consequence of increasing formation
of oxygen radicals. In mammalian extracellular fluids, a heme-binding
protein called hemopexin has been shown to diminish the effectiveness
of heme as a pro-oxidant
(4, 5) . Hemopexin also is
involved in heme transport in vertebrate plasma
(6) . The
question of how blood-feeding insects deal with the large amounts of
heme in their diet has received very little attention.
Here we
describe the isolation and characterization of a heme-binding protein
from the blood-sucking insect, Rhodnius prolixus.
Insects Insects were taken from a colony of R. prolixusmaintained at 28 °C and 70% relative humidity. Normal mated
females were fed on rabbit blood at 2-week intervals. Hemolymph and Oocytes Four to 6 days after a meal, hemolymph was collected in the presence of
phenylthiourea (30-130 µg/ml), 5 mM EDTA, and a
mixture of protease inhibitors prepared in 0.15 M NaCl, with
final concentrations of 0.05 mg/ml of soybean trypsin inhibitor,
leupeptin, lima bean trypsin inhibitor and antipain, and 1 mM
benzamidine. On the same day, chorionated oocytes were dissected and
washed with ice-cold 0.15 M NaCl in order to remove ovarian
debris prior to homogenization. Oocytes were homogenized in a
Potter-Elvehjem homogenizer in the presence of the same mixture of
protease inhibitors, buffered with 20 mM Tris-HCl, pH 7.0,
(approximately 500 oocytes to 1 ml), and centrifuged at room
temperature in a microcentrifuge at 11,000
Molecular weights of
polypeptides were measured by SDS-PAGE using the following protein
standards: myosin (205 kDa),
Purified RHBP (0.5 mg) was adsorbed to a
piece of nitrocellulose filter that, after saturation with a 5%
solution of powdered milk, was used as an affinity matrix to isolate
anti-RHBP IgG. The procedure has been described in detail elsewhere
(8) . This IgG was used to perform Western blots according to
Towbin et al.(9) . Amino Acid Analysis RHBP isolated from oocytes (4 nmol) was hydrolyzed for 22 h at 110
°C in 0.5 ml constant boiling 6 N HCl containing 0.01%
phenol in an evacuated sealed tube. Amino acid analysis was carried by
the method of Spackman et al.(10) using an automatic
instrument
(11) . No corrections were made for losses during
acid hydrolysis. Hydrolysis with lithium hydroxide
(12) was
used for determination of tryptophan. NH
High performance liquid chromatography
gel filtration was carried out using a TSK-125 column and pre-column
equilibrated with 0.1 M sodium phosphate, pH 6.8, and eluted
at 0.8 ml/min using a LDC series 4000 pump and detector (set at 412 nm)
and a CI-10 integrator. Heme Binding Assay Binding of heme to RHBP was monitored by measuring the absorbance of the
Soret band at 412 nm while progressively adding a solution of 1
mM hemin in 0.1 M KOH
(16) . The absorbance
was plotted against the molar ratio of added hemin to polypeptide
( M
The pigment that gives Rhodniuseggs and
hemolymph their characteristic pink color in both cases copurified
throughout all protein isolation steps with a polypeptide of 15 kDa
(Fig. 1, A and B).
The absorption spectra
of the proteins from oocyte and hemolymph are typical of a
heme-containing protein (Fig. 6, A and B). The
protein was isolated from both sources in the ferric state ( solid
line), and spectra determined on fresh crude oocyte extracts and
hemolymph also showed the pattern typical of the oxidized protein (data
not shown). Reduction with sodium dithionite ( dashed line)
changed both intensity and position of the major
Our results show that the blood-sucking hemipteran R.
prolixussynthesizes a unique heme-binding protein of 15
kDa, not previously described. To our knowledge, this is the first
description of a heme-binding protein in a blood-feeding insect. In a
classical report, Wigglesworth
(17) showed that the pigment
present in Rhodniushemolymph and eggs was associated
with a protein that he believed was produced by partial digestion of
hemoglobin from the vertebrate host. However, on the basis of the
present work, this can be ruled out, since specific antibodies against
RHBP do not cross-react with rabbit hemoglobin (Fig. 4). The
conclusion that RHBP is a different protein, synthesized by the insect
itself, is also supported by a clearly distinct amino acid composition
( and Ref. 18 and 19) and by its NH
Titration of the apoprotein with
hemin shows that there is only one binding site/polypeptide chain
(Fig. 7). Addition of hemin to the native protein purified from
the hemolymph, but not from the oocyte, is immediately followed by its
binding to the protein (Fig. 8), indicating that the protein in
the hemolymph is not saturated. The possibility that the heme-binding
site in the purified protein is an artifact generated during the
isolation procedure can be excluded, since titration of crude hemolymph
with hemin gave similar results (data not shown).
Vertebrate plasma
also has a heme-binding protein, hemopexin
(22) . Hemopexin and
RHBP share absorption spectra in the visible range typical of
b-type cytochromes ( Fig. 6and Refs. 20, 21, and 23).
RHBP (Fig. 2) is only one-fourth the size of hemopexin (60 kDa)
(22) . Limited proteolysis of hemopexin produces a peptide that
can still bind heme
(24) . However, the CD spectrum of the
insect heme-binding protein (Fig. 3) reveals a highly helical
secondary structure instead of the 95% random-coil pattern displayed by
hemopexin
(25) . The amino acid sequence at the NH
In the oocyte, RHBP is found inside the yolk platelets (data not
shown), which are organelles specialized in storing materials for
growth of the embryo
(26) . Therefore, in the egg RHBP may be a
source of heme or iron for insect embryogenesis, as the protein is
accumulated in the oocyte in significant quantities, as indicated by
the strong 15 kDa band in the crude oocyte extract
(Fig. 1 A).
Hemin is a powerful generator of free
radical reduction products of dioxygen that are capable of causing
biological injury through peroxidation of lipids, proteins and DNA
(3-5 and 27). The antioxidant role of RHBP is investigated in the
following article
(28) .
Data are reported for a 22-h acid hydrolysis and amino acid analysis
in duplicate. The integral molar values, residues/mol, are given in
parenthesis. No corrections have been made for losses during acid
hydrolysis. The minimal chemical molecular weight calculated for 132
residues/mol is 16,125.
We express our gratitude to Dr. Martha M. Sorenson and
to Dr. John H. Law for a critical reading of the manuscript and to
Rosane O. M. M. Costa, José S. Lima Jnior, José F. Souza
Neto, and Sebastiana S. Santos for excellent technical assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-helical structure which binds non-covalently one
heme/polypeptide chain. This RHBP is not produced by limited
degradation of hemoglobin from the vertebrate host, since specific
polyclonal antibodies against it do not cross-react with rabbit
hemoglobin, and since it differs from hemoglobin in having a distinct
amino-acid composition and NH
-terminal sequence. The
spectrum of the dithionite-reduced protein has peaks at 426, 530, and
559 nm and resembles that of a b-type cytochrome.
g for 5
min. The floating lipids and the pellet were discarded, and the clear
supernatant was used as the crude oocyte extract for protein
purification. Purification of RHBP
(
)
From Oocytes
Solid ammonium sulfate was
added to bring the oocyte extract to 45% saturation, and the suspension
was gently stirred for 20 min at 4 °C. After centrifugation at
11,000 g for 10 min, the precipitate was discarded,
and the supernatant was brought to 60% saturation. This new precipitate
was then washed twice with a 60% saturated ammonium sulfate solution
and then was back-extracted by resuspending in a 45% saturated solution
and centrifuging. The pellet was discarded and the supernatant was
dialyzed against 0.15 M NaCl, 10 mM Tris-HCl, pH 7.0,
and applied to a column of Sephadex G-200 (2.5
55 cm)
equilibrated with the same solution. Protein content of fractions was
measured by the absorbance at 280 nm. The colored fractions containing
RHBP were pooled, dialyzed against deionized water, and lyophilized.
From Hemolymph
Hemolymph (approximately 3 ml) was
diluted to 5 ml with phosphate-buffered saline (0.15 M NaCl,
0.1 M sodium phosphate, pH 7.0) and 1.25 g of KBr was added.
The solution was centrifuged at 80,000 g for 20 h at 4
°C. The fractions at the bottom of the tube were collected and
dialyzed against deionized water until an abundant precipitate had
formed. The solution was then centrifuged at 11,000
g for 10 min at 4 °C. The supernatant was brought to 10
mM with Tris base and applied to a column (1.5
18 cm)
of DEAE-Toyopearl, equilibrated with 10 mM Tris-HCl, pH 8.4.
The column was first washed with 20 ml of the same buffer and then
eluted with an NaCl gradient (0-100 mM). The fractions
containing RHBP were pooled and applied to a Sephadex G-75 column (1.5
80 cm) equilibrated with 0.15 M NaCl, 10 mM
Tris-HCl, pH 7.0. Fractions containing the RHBP were pooled, dialyzed
against deionized water, and lyophilized. Polyacrylamide Gel Electrophoresis Polyacrylamide gradient (6-22.5%) gels (10
10 cm
1
mm) were run in the presence of SDS
(7) at a constant current
of 22 mA. Gels were stained with Coomassie Brilliant Blue R and
destained with 7% acetic acid in 40% methanol.
-galactosidase (116 kDa),
phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45
kDa), glyceraldehyde-3-phosphate dehydrogenase (36 kDa), carbonic
anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20
kDa), and
-lactalbumin (14 kDa). Antibodies and Western Blot RHBP (1 mg) purified from oocytes was emulsified with an equal volume of
Freund's adjuvant and injected subcutaneously into the back of a
rabbit. Boosters of 0.5-1.0 mg of protein were injected at
2-month intervals, and blood was collected from an ear vein 3 months
after the first interval.
-terminal Sequence Purified RHBP (100 pmol) was submitted twice to automatic Edman
degradation using a liquid phase sequencer (Porton PI 2020/2090).
Phenylthiohydanthoine amino acids were identified using a Hewlett
Packard HPLC with a reverse phase AminoQuant analytical column (2.1
250 mm). The initial and repetitive yields obtained were 52 and
96%, respectively. Sequence comparisons were performed according to
Devereux et al.(13) . Visible Absorption Spectra Visible spectroscopy experiments were carried out in 60 mM
NaCl, 20 mM Tris-HCl, pH 8.0, on a Cary 100 spectrophotometer.
The reduced form of RHBP was obtained by addition of small amounts of
sodium dithionite. Reoxidation was achieved by progressive addition of
potassium ferricyanide until dithionite consumption and reappearance of
the ferric spectrum. Heme Extraction The heme group of RHBP was extracted by a modification of the
acetone-HCl method
(14) , using a final concentration of HCl of
25 mM and washing the protein pellet until total extraction of
heme. Circular Dichroism The circular dichroism spectrum between 190 and 250 nm was obtained with
the protein from oocytes, in an Aviv 60 DS circular dichroism
spectropolarimeter (Aviv Associates, Inc., Lakewood, NJ) and a 0.05-mm
quartz cell. Data were analyzed according to Chang et al.(15) . Five spectra were averaged and smoothed. Errors in
the spectra were insignificant in comparison with errors in protein
concentration and pipetteting. Gel Filtration The apparent molecular weight of RHBP from both the oocytes and the
hemolymph was determined with the Sephadex G-75 column used during
isolation from the hemolymph. The column was calibrated with proteins
of known molecular weight.
= 15,000), and the hemin necessary to
fully form the holoprotein was determined from the break in the curve.
Figure 1:
Summary of RHBP
purification from oocytes and hemolymph analyzed by SDS-PAGE.
A, from oocytes: 1, crude oocyte extract; 2,
after ammonium sulfate precipitation; 3, after Sephadex G-200
chromatography. Molecular weights at the left are vitellin apoproteins.
B, from hemolymph: 1, crude hemolymph; 2,
subnatant from KBr density centrifugation; 3, supernatant
after precipitation against water; 4, after DEAE-Toyopearl
column; 5, after Sephadex G-75
chromatography.
Both the hemolymph and
oocyte proteins are monomeric, as indicated by an apparent molecular
weight of 12,400 for the native protein as estimated by gel filtration
chromatography (Fig. 2) and 15,000 by SDS-PAGE. The amino acid
composition corresponding to this size is shown in .
Figure 2:
Native molecular weight determination. The
molecular weight of RHBP from both oocytes and hemolymph
( arrow) was determined using a Sephadex G-75 column. The
following proteins were used as standards: albumin ( A),
ovalbumin ( O), carbonic anhydrase ( C), cytochrome
c ( Ci) , and aprotinin ( Ap).
Ve;, elution volume; V, exclusion
volume.
Analysis of the secondary structure by circular dichroism
(Fig. 3) shows a mean residue ellipticity of -22,635
degcm
dmol
at 222 nm. The
best estimate indicates a high proportion of
-helix (75%).
Figure 3:
Circular dichroism spectrum of RHBP. RHBP
isolated from oocytes was used at a concentration of 0.1 mg/ml at 30
°C in 10 mM sodium phosphate buffer, pH
7.0.
In
order to determine whether the oocyte and hemolymph proteins are
related and whether they are derived from ingested proteins or
synthesized by the insect itself, antibodies against the purified
oocyte protein were raised in rabbits. The specific IgGs were purified
as described under ``Experimental Procedures'' and used in a
Western blot. Fig. 4shows that a 15 kDa band is specifically
recognized in the hemolymph of females and oocytes. Interestingly,
although hemolymph from fourth stage larvae is not as strongly colored
as that from adult females, the same 15-kDa band is seen in the Western
blot (Fig. 4, lane 2), indicating that the protein is
not exclusively related to oogenesis.
Figure 4:
Western
blot of hemolymph and oocytes. Affinity-purified IgG against RHBP was
used for a Western blot (see ``Experimental Procedures'').
Samples were run on a 5-15% SDS-PAGE gel prior to electrophoretic
transfer to a nitrocellulose membrane. Shown are: 1, crude
oocyte extract; 2, fourth-stage larvae hemolymph; 3,
adult female hemolymph; 4, rabbit hemoglobin; and 5,
RHBP purified from oocytes.
Determination of the
NH-terminal amino acid sequence by automated Edman
degradation revealed total identity of the oocyte and hemolymph
proteins in the first 36 residues (Fig. 5).
Figure 5:
NH-terminal amino acid
sequence. The NH
-terminal primary structure of RHBP from
both oocytes and hemolymph was determined by automated Edman
degradation and found to be identical.
Searching protein
data bases (SWISS-PROT, GenBank, and EMBL) with this sequence did not
indicate any significant similarity to already known polypeptides. This
result clearly establishes that the protein is not derived from dietary
hemoglobin, as suggested previously
(17) , but is instead
synthesized by the insect itself. The same conclusion is reinforced by
the amino acid composition (), which is distinct from
vertebrate hemoglobin
(18, 19) and by the lack of
immunological cross-reaction between rabbit hemoglobin and RHBP, as
revealed by the Western blot (Fig. 4).
-peak, which
shifted from 412 to 426 nm, and produced the characteristic
- and
-bands at 559 and 530 nm, respectively. This low spin state
spectrum closely resembles that of b-type cytochrome, which
are characterized by two histidine axial ligands
(20, 21) . The absence of peaks at the 620 and 695 nm
regions of the oxidized spectrum (high spin and methionine axial ligand
indicators, respectively) also are similar to b-type
cytochromes (data not shown). Nevertheless, more direct information on
heme axial ligands ( e.g. magnetic CD spectra) is still
lacking. The alkaline pyridine derivative obtained in 0.2 N
NaOH and 20% pyridine gives, after reduction with dithionite, a typical
protoheme band at 558 nm (data not shown).
Figure 6:
Visible absorption spectra of oxidized and
dithionite-reduced RHBP. Spectra from protein purified from oocytes
( A) or hemolymph ( B) were recorded in 60 mM
NaCl, 20 mM Tris-HCl, pH 8.4. Shown are: -, native
protein (no addition); - - - - , dithionite-reduced;
, reoxidized by potassium
ferricyanide.
In common with other
proteins that exhibit a b-type cytochrome absorption spectrum,
the heme group can be extracted by the acetone-HCl method
(20) ,
and the resulting apoprotein can be titrated by adding back the hemin
(Fig. 7). As hemin bound to the protein has a higher extinction
coefficient at 412 nm than free hemin, the saturation of the apoprotein
is indicated by a break in the straight line on the plot of absorbance
at 412 nm against the amount of hemin added to the medium. The two
lines intersect at a hemin-to-polypeptide ratio of approximately 1:1,
demonstrating that only one heme group is bound by each polypeptide.
Figure 7:
Heme-RHBP reconstitution. The heme group
of RHBP from hemolymph was extracted by the acetone-HCl method. The
apoprotein was diluted in 0.15 M NaCl, 20 mM HCl, pH
7.5, and titrated by addition of hemin and measurement of the
absorbance at the Soret band (412 nm).
Although the native RHBP purified from hemolymph already has some
heme bound to it, addition of hemin also produces a break in the plot
(Fig. 8 A), revealing that the protein is not saturated
and that about half of the protein molecules in the hemolymph are
apoproteins. A different result is obtained with the oocyte protein,
where addition of heme shows no available heme-binding sites,
indicating that in the oocyte RHBP is fully saturated
(Fig. 8 A). As a control, aliquots from the experiment
shown in Fig. 8 A were applied to a gel filtration
TSK-125 HPLC column while monitoring the absorbance of the eluant at
412 nm (Fig. 8 B) and observing the increase in the
height of the peak. Saturation was obtained with the same amount of
added hemin as in the previous experiment.
Figure 8:
Binding of heme to the hemolymph protein.
Association of heme with the protein was measured by the increase in
absorbance at the Soret band (412 nm) as in the previous figure. Shown
are: A, titration of RHBP purified from hemolymph () and
oocytes (
); B, gel permeation high performance liquid
chromatography of native hemolymph RHBP after different additions of
hemin. Numbers inside the figure indicate ratios of added
hemin to polypeptide.
-terminal
amino acid sequence (Fig. 5), which does not indicate significant
homology to any known protein.
terminus of RHBP is also unique, reinforcing the same conclusion.
These observations suggest an independent evolutionary origin for RHBP.
Table:
Amino acid composition of heme RHBP from oocytes
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.