Biochemical variability between venoms from different honey-bee (Apis mellifera) races

Camp. Biochem. Physiol. Vol. 106C,No. 2. pp. 423427, 1993

0742-8413193$6.00 + 0.00 Pergamon Press Ltd

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BIOCHEMICAL DIFFERENT

VARIABILITY BETWEEN VENOMS FROM HONEY-BEE (APlS ~~~~~~~~~) RACES M. S. PALMA and M. R. BROCHETTO-BRAGA

Departmentof Biology, Biosciences Institute--UNESP,

CEIS/CEVAP, Rio Claro, SP, 13506-900, Brazil

(Received 22 April 1993; accepted for pubiication

Abstract-l.

The

comparison

11 June 1993)

profiles of venoms from sting and Africanized honey-bees in Sephadex

of molecular exclusion cromatography

apparatuses of Apis melltjkra ligustica, Apis mellifra

adansonii

G-100 revealed both qualitative and quantitative differences. 2. The venoms from A.m. ligwtica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former. 3. The polypeptides with IU, in the range from 100,000to 7500 Da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. udansonii and Af~~nized honey-bees, while the peptidic fraction with M, range from 4100 to 2000 Da corresponds to 11.4%,32.4% and 10.2% of venom protein, respectively.

Despite early knowledge about the nature of the venom from honey-bees, it was in the 19th Century that it started to attract scientific interest (Langer, 1897), but at this time the studies simply showed that the venom was a complex mixture of substances. It was only in the fifties that Neumann et al. (1952) demonstrated that the proteic and peptidic components were associated with biological activity of honey-bee venom. When Benton et al. (1963) developed an apparatus for venom collection, it became possible to obtain large quantities of relatively pure venom. Thus, some groups of researchers became involved in the characterization of honey-bee venom and contributed to elucidation both of composition and mechanisms of actions of the individual components (Habermann, 1972; O’Connor and Peck, 1978; Banks et al., 1979). Although the biochemistry of this venom is well known (Banks et al., 1981; Banks and Shi~lini, 1986; Dotimas and Hider, 1987; Habermann, 1972; Mello, 1970), there are few comparative studies between the different honey-bee races. Shipman and Vick (1977) described some chromatographic differences between the venoms of European (EHB) and Africanized honey-bee (AHB); Schumacher et af. (1990) demonstrated the existence of electrophoretic differences between venoms of the EHB and AHB. Thus, it seems possible for some biochemical differences to exist in the venom composition, at the level of proteins and peptides, between the different subspecies of A. mell$era, between genetically heterogeneous colonies of AHB and between both groups. However, the previous studies do not take into account some variables which may frequently influence the qualitative and/or quantitative chemistry of

honey-bee venom: (1) the age of worker bees (Owen, 1978; Owen et al., 1990; Bachmayer er al., 1972; Owen and Bridges, 1982); (2) the date of sample collection is important, because there are seasoncorrelated variations (Owen and Sloley, 1988); and (3) the techniques of venom collection and storage also may influence the composition (Piek, 1986). Thus, the aim of present work was to characterize some biochemical differences between the venoms from different races of Apis mellifera through comparative analysis of molecular exclusion chromatography profiles, maintaining, under controlled conditions, the sources of variability mentioned above.

MATERIALSANDMETHODS Collection of bees and venom isolation Colonies of A.m. ligustica, A.m. adansonii and AHB were maintains in the apiary of the Biosciences Institute at the University Campus of Rio Claro, SHo Paulo State, southeast of Brazil. The colonies of A.m. adansonii were descendants of queens originally brought from Ghana, Africa. To ensure the genetic homogeneity of each race, all colonies used in this study were descendants of queens inseminated by instrumentation, using semen from a drone of the same maternal race. The age of honey-bees was determined by marking the thorax of newly emerged individuals, premiting to follow them. Honey-bee workers (HBW) aged between 25 and 30 days were captured with an insect net, near the entrance of the colonies of each race, on the same day. The captured bees were immediately cooled on ice, frozen and stored at - 16°C until dissection.

423

M. S. PALMA and M. R.

424

BROCHEITO-BRAGA MOLECULAR

Venom sacs from 600 HBW from each colony were removed from the stinging apparatus by pulling with forceps and cutting with microscissors under a without contamination of exstereomicroscope, traneous tissues. The sacs were then carefully washed in a small volume of isotonic solution, thawed and centrifuged at 12,OOOg,15 min at 4”C, and the supernatant was freeze dried. The venoms obtained by this procedure were kept at - 16°C in amber flasks until be used.

EXCLUSION

CHROMATOGRAPHIRS

The freeze dried venom was reconstituted in 10 mM ammonium acetate pH 6.8; 26 mg of proteins (13 mg/ml) were applied to a Sephadex G- 100 column (52 x 2 cm) and 13 mg (13 mg/ml) to a Sephadex G-15 column (30 x 1 cm), previously equilibrated with the same solution of reconstitution. The elutions were carried out at lOml/hr, collecting fractions of 3 ml. The column eluant was monitored by spectrophotometric determination of protein and by optical density at 280 nm.

Protein determination

MOLECULAR

Protein concentrations were determined by the method of Lowry et al. (1951), using bovine serum albumine as standard.

WEIGHT ESTIMATION

The native molecular weight of each eluted fraction, was estimated using the gel permeation columns

0.700 0.600 0.300 0.400 0.300 0.200 0.100 0.000 0.600

AFRICANIZEO HONEY BEE 150

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0.500

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:

:

60

0.600

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6 0.400

88 =*

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22

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0.300

22

2 20 0.200

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,

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,

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TO 60

VOLUME

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.

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100 110 120

ELUTION

(ml )

Fig. I. Fractionation profiles for gel permeation of crude venom from sting apparatuses of different honey-bee races, on Sephadex G-100 column (52 x 2 cm) with 10 mM ammonium acetate pH 6.8 at a Row rate of 10 ml/hr. Fraction size: 3 ml.

425

Biochemical variability of honey-bee venoms

r-

SW-

A. m.

adanseeii

- 0.600 - o.!$oo

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450 ”

- 0.800 - 0.200

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AFRICANI HONEY BE2

o&O0

0.500

“0

20

40

60

80

100 120 140 160

180 200

220 240 260 2B0 300 320 VOtUME

Fig. 2. Fractionation profiles for gel permeation

OF

ELUTION

340

360

(ml)

of crude venom from sting apparatuses of different

honey-bee races, on Sephadex G-15 column (30 x 1cm) with 10mM ammonium acetate pH 6.8 at a Row rate of lOml/hr. Fraction size: 3 ml.

in the same conditions described above. The column of Sephadex G-100 was previously calibrated with the following molecular weight markers: bovine serum albumin, 67,000; ovalbumin, 43,000; trypsin, 22,500; lisozyme, 16,600 and aprotinin, 6500. The column of Sephadex G-15 was calibrated with the following molecular weight markers: corticotropin, 4600; mellitin, 2840 and bacitracin, 1800. RESULTS The gel permeation profiles of crude venoms in Sephadex G-100 and Sephadex G-l 5 columns are shown in Figs 1 and 2, respectively. The profiles of f~ctionation in Sephadex G-100 (Fig. l), revealed a total of 21 protein peaks with h4, from 7500 to 100,000 Da. In these profiles, the UV absorbance at 280 run is proportional to protein concentrations only

for the peaks 1 and 2, being different for each race. In the final part of each chromatogram, there are pronounced UV peaks, with very low protein concentrations. Most protein peaks presented very low absorbance at 280 mm. The M, value and the percentual of occurrence of each protein pool eluted in these chromatographies are shown in Table 1. The venoms from A.m. adansonii, A.m. ligwica and AHB presented, respectively, 19, 17 and 13 protein peaks. A.m. adansonii was characterized by the presence of three peaks unique for this race; no. 8, 12 and 16 (M, 32,000, 22,000 and 16,000); A.m. Iigusticu was characterized by the presence of one unique peak: No. 1 (M, l~,~), while AHB was characterized by the absence of eight peaks (No. i,4, 6,8,10,12,16 and 19) and presented no peak unique for the race.

M. S. PALMA and M. R. BROCHETTO-BRAGA

426

Table 1. The M, values and the percentual of occurrence of each protein peak eluted from gel permeation chromatogaphy of crude venoms from sting apparatus of different honey-bee races, in Sephadex G-106 columns A.m.

Peak I 2 3 4 5 6 1 8 9 10 11 12 I3 14 15 16 17 18 19 20 21

% of

M. 100,000 87,000 66,000 56,009 51,000 43,000 38,000 32,000 29,000 27,000 24,000 22,090 20,900 18,000 17,Gw 16,900 15,009 12,000 10,500 8700 7500 total venom

adansonrr

20.1 4.3 6.0 3.9 3.1 3.0 1.7 2.3 2.0 25 I6 1.5

A.m.

thstrca 25.0 6.0 3.0

African&d honev-bee

5.0 2.0 -

;I: 2:5 2.4 -

1.0 -

2.0 1.8 2.0 -

0.9 -

3.0 -

4.7 4.1 3.0 10.3 2.3

::t 3.8 1.8 1.6 I .4 1.6

2.0 2.2 1.5 I .4 1.0 0.7

66.6

62.0

I.7 -

2.5 -

-

17.0 14.2 68.7

The profiles of fractionation in Sephadex G-15 (Fig. 2) revealed a total of 1I peptide peaks with M, from 2000 to 4100 Da. The UV profiles are similar for the three honey-bee races. The M, value and the percentage of occurrence of each peak eluted in these chromatographies are shown in Table 2. The venoms of A.m. adansonii, A.m. ligustica and AHB presented, respectively, 9, 8 and 10 peptide peaks. The venoms of A.m. adansonii and AHB do not present characteristic peptide peaks; however, A.m. ~i~tica presented one peak unique for the race: No. 31 (MW 2.450). In addition to qualitative aspects, Tables 1 and 2 also show the occurrence of quantitative differences. Thus, Table 1 reveals that the polypeptides with M, in the range lOO,OOO-7500Da correspond to 66.6%, 62.0% and 68.7% of total proteins from the venom of sting apparatuses of A.m. adansonii, A.m. ligustica and AHB, respectively. In addition to this, Table 2 shows that the peptides with M, in the range 4100-2000 Da correspond to 32.4%, 11.4% and Table 2. The M, values and percentual of occurrence of each peptide peak eluted from gel permeation chromatography of crude venoms from sting apparatus of different honeybee races, in Sephadex G- 15 columns A.m.

ad~nso~j~

A.m.

Iigustica

African&d honey-bee

Peak

M.W.

22 23 24 2.5

4100 3900 3800 3600

9.0 2.8 1.4 1.7

4.6 0.6 1.9

1.6 I.1 0.2 0.8

c

3350 3250

G

0.4 -

0.5 0.1

:i 30 31 32

2800 3000 2600 2450 2oOil

2.0 5.0 6.5

1.1 I.5 0.4 0.9

0.1 0.4 1.1 3.7

32.4

11.4

10 2

% of total venom

10.2% of protein fraction from the venoms of A.m. adansonii, A.m. ligustica and AHB. In addition to this, Tables I and 2 also show quantitative differences for individual components, like the high relative concentrations of the peaks 2,4, 22, 30 and 32 (A4, 87,000, 56,000, 4100, 2600 and 2000) in the venom of A.m. adunsonii and of the peaks 17, 20 and 21 (MC 15,000, 8700 and 7500) in the venom of AHB. The first peak observed in the cromatographies performed on Sephadex G-15 (Fig. 2) was eluted in the void volume of the column; thus, it represents the proteic/peptidic material with iw, > 4100 Da, excluded from fractionation. Other quantitative differences may also be observed, which seems to be of minor importance. DISCUSSION

Nelson et al. (1990), comparing the venoms of EHB and AHB, observed no differences of composition; however, Schumacher et al. (1990, 1992) demonstrated the occurrence of a lower content of mellitin and a higher content of phospholipase A, in the venom of AHB, when compared to EHB. Gwen et al. (1990) observed a lower content of hyluronidase in the venom of A.m. adansonii than in EHB. In spite of these observations which suggest the occurrence of biochemical va~ability between venoms from different honey-bee races, variables do exist that influence the chemistry of these venoms, which were not taken into consideration in the previous investigations. Thus, in the present work it was decided to carefully characterize the occurrence of some biochemical differences between the venoms of different races of Apis mellifera. In order to do this, the sampling of workers was rigorously carried out, by controlling the age of individuals, the date (season) and technique of collection and by ensuring both the genetic homogeneity of each colony used and that all honey-bee races were reared under the same environmental conditions. Maintaining these controlled variables, the present study demonstrated the existence of qualitative and quantitative differences between the venoms of sting apparatus from A.m. ligustica, A.m. adansonii and AHG. The M, ranges of 100,00&7500 and 410%2000 were chosen for fractionation because they comprise the enzymes and the majority of the known peptides from honey-bee venom. If one considers that the fractionation of venoms was performed by a molecular exclusion process, it must be remembered that each peak of proteins and/or peptides may comprise several different components with the same apparent Mr Thus, it is possible that new qualitative differences will appear after re-fractionation of each peak, by another separation process. The percentage of polypeptides with IM,3 7500 Da is similar between the three venoms (Table 1);

Biochemical variability of honey-bee venoms

427

perspective study with emphasis on the clinical aspects. Clin. Allergy II, 311-327. Hider R. C. (1988) Honey bee venom: a rich source of pharrnacollogically active peptides. Endeaoour 12(2), ~5. Langer J. (1987) Uher das Gift unserer honigbiene. Arch.

however the levels of peptides in the M, range 4100-2000 Da seems to be 3-fold higher in the venom of A.m. adansonii than in the venoms from A.m. ligustica and AHB (Table 2). The peptide fraction of h&rev-bee venom contains imnortant bioloeicallv active components like mellitin, Bpamin, MC6 pe& tide, secapin, tertiapin and procamin (Dotimas and Hider, 1987; Hider, 1988). Thus, it is probable that the venom of A.m. adansonii is richest in these components than A.m. ligustica and AHB. Although the fractionation by molecular exclusion is not a high resolution technique for protein and/or peptide separation, there are enough differences, both aualitative and auantitative. between the venoms of sting apparatus from A.m. adansonii, A.m. ligustica and AHB, that the chromatographies in Sephadex G-100 and G-15 do offer an opportunity for identification of each race.

O’Connor R. and Peck M. L. (1978) Venoms of the Apidae.

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_

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Shipman W. H. and Vick J. A. (1977) Studies of Brazilian bee venom. Cutis 19, 802-804.