INFECTION AND IMMUNITY, Dec. 1974, p. 1412-1419Copyright © 1974 American Society for Microbiology

Vol. 10, No. 6Printed in U.S.A.

Immunological Studies of Brown Recluse Spider VenomKLAUS D. ELGERT,I MILTON A. ROSS, BENEDICT J. CAMPBELL, AND JAMES T. BARRETTDepartments of Microbiology and Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65201

Received for publication 15 July 1974

Polyacrylamide gel electrophoresis of Loxosceles reciusa venom demonstratedthat only one of seven or eight major (plus three or four minor) proteincomponents caused necrosis in guinea pig skin. Sephadex gel filtration separatedthe venom into three major peaks, the second peak of' which contained thedermonecrotic activity. Hyperimmunization of rabbits with increasing doses ofvenom from L. reclusa produced potent precipitating antisera, and the rabbitsbecame resistant to lesion development. Ouchterlony-type immunodiffusion andimmunoelectrophoretic studies revealed six to seven distinct precipitation lines,one of which stained intensely for esterase activity. Immunohistochemicaltechniques failed to detect any protease, lipase, catalase, acid phosphatase,alkaline phosphatase, or amylase activity in the venom. The spreading activity ofrecluse spider venom in guinea pig skin was inhibited as much as 71% byantivenom. Venom preincubated with antivenom was unable to incite lesions inguinea pig skin. Passive immunization of' guinea pigs 18 h before an injection ofvenom conferred venom resistance upon the animals. Local injections ofantivenom immediately after intradermal injections of venom markedly reducedthe dermal lesion. Heparin reduced the local and systemic effects of venom whenpreincubated with whole venom or when administered systemically before anintradermal injection of venom. Treatment of whole venom with the chelatingagent ethylenediaminetetraacetate did not inhibit its necrotic activity. Transferstudies from a 24-h lesion indicated that the necrotic activity was localized andremained active in tissue for at least 24 h but not for 5 days. No lesions developedwhen high concentrations of venom were intradermally injected into the skin ofsacrificed guinea pigs, indicating that an interaction of body constituents andvenom is essential for the development of a lesion.

The species Loxosceles reclusa was not associ-ated with necrotic arachnidism until 1958, whenAtkins et al. (1) demonstrated that bites of L.reclusa, the brown recluse spider, produced anextensive, gangrenous lesion in rabbits.Though there have been numerous reports

and case studies of' persons bitten by brownrecluse spiders (6, 7, 13, 15), fundamentalimmunological studies of' the venom are few.Denny et al. (4) demonstrated an in vivo and invitro hemotoxic effect of L. reclusa venom thatwas reduced approximately 70% in activity byrabbit antivenom. Unfortunately, a normal rab-bit serum control was not included in thisexperiment. Circulating antivenom was demon-strated in the serum of dogs and rabbits surviv-ing spider bites, though antibodies were notdetected after a single bite (4). Immunodiffu-sion studies of three species of Loxosceles (18)revealed that each of the venoms contained

'Present address: Department of Biology, Virginia Polv-technic Institute and State University, Blacksburg, Va.24061.

three antigen components detectable by theOuchterlony technique and four by immuno-electrophoresis. The immunoelectrophoreticpatterns of the venom of the three species weredistinctly different.

Before immunoprophylaxis and immuno-therapeusis of brown recluse spider bites canbecome a reality, further immunological andbiochemical understanding of the behavior oftoxins in the venom of this spider is essential.Such is the subject of this report.

MATERIALS AND METHODSCollection of spider venom. To insure an optimal

harvest of venom, food was withheld from adultspecimens of L. reclusa for 2 weeks before venomcollection. The paired venom glands of anesthetizedspiders were exposed by microdissection, after whichpure venom was expressed into an appropriateamount of 0.1 M phosphate-buffered saline (PBS) bygently pressing the glands with forceps. The proteinconcentration of venom solutions was determined bythe method of Lowry et al. (14).

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BROWN RECLUSE SPIDER VENOM

Gel electrophoresis. Polyacrylamide gel electro-phoresis was performed on 210 ,ug of venom protein byusing the standard 7.5% acrylamide gel at pH 8.9 in0.05 M tris(hydroxymethyl)aminomethane-glycinebuffer. A Bio-Rad model 150 gel electrophoresis as-sembly (Bio-Rad Laboratories, Richmond, Calif.) wasused at 3 mA per tube. The gel was stained withCoomassie blue for 2 h and electrophoretically de-stained. A companion gel also containing 210 ,ug ofvenom protein but not stained was sliced at 3-mmintervals, and each slice was eluted into 0.4 ml of PBSfor 24 h at 4 C, after which a 0.1-ml portion wasinjected intradermally into guinea pigs. A slice takenin advance of the ion front, which contained noprotein, served as a control on the effect of polyacryl-amide gel on guinea pig skin.Column preparation and elution. Gel filtration

experiments were conducted by using a column (1.5by 30 cm) (Pharmacia Fine Chemicals, Piscataway,N.J.) packed with medium-mesh Sephadex G-75equilibrated with PBS. A 1.0-ml sample of PBScontaining approximately 800 jg of venom proteinwas applied to the column. Fractions of approxi-mately 1.0 ml were collected, and their absorbancewas monitored at 280 nm. All Sephadex filtrationexperiments were performed at 4 C.

Samples of 0.1 ml were taken from the tube in eachabsorption peak that had the greatest optical density,and these were injected intradermally into guinea pigsas was a sample of venom-free buffer.

Preparation of antivenom and immunodiffusionstudies. Hyperimmune antivenom was obtained byrepeated intradermal injection of whole venom inincreasing quantity into rabbits at intervals of a fewdays and over a period of approximately 10 weeks.The initial injection consisted of 14 jg of venomprotein, was increased to 47 jg, and then was held at alevel of 30 Ag until the rabbits proved refractory tolesion development. Eight days after the final venominjection, serum was collected by heart puncture.

Antibodies to venom components were demon-strated by the Ouchterlony gel diffusion technique.The immunodiffusion plates were prepared by using1% Noble agar in barbital buffer, pH 8.6, ionicstrength 0.05. dissolved in 0.85% saline. The plateswere incubated at room temperature for 48 h.

Portions of peak absorption tubes 20, 25. 34, 60, and70 from the Sephadex-fractionated and whole venomwere diffused and reacted against antivenom (Fein-berg agar gel cutter, Shandon Punch. Colab Products,Chicago Heights, Ill.). The center well was filled withantiserum, and venom or venom fractions were ar-ranged to alternate in the peripheral wells.The immunoelectrophoresis slides were prepared

with the same Noble agar used in the Ouchterlonytechnique. An LKB 6800-1 Immunophor StandardSet apparatus and power supply (LKB Producter AB,Stockholm, Sweden) were used to provide a current ofapproximately 10 V/cm for 1 h, after which antiserumwas placed in the trough. Precipitation bands werestained with amido black.The lipid staining procedure of Uriel (22) was

applied to Ouchterlony immunodiffusion plates thathad been incubated for 72 h at room temperature. The

method used to stain for esterase activity in immuno-precipitates was that of Clausen (3) modified afterUriel (21). Slides from the immunodiffusion andimmunoelectrophoresis studies were also stained byhistochemical procedures (16) to determine whetherprotease (17), lipase, catalase, alkaline and acidphosphatases, or amylase (22) activity was present.Venom neutralization. To determine whether an-

tivenom would neutralize the venom's necrotic effect,experiments similar to those described earlier (23)were performed. Varying concentrations of venom in0.1 ml of saline were mixed with 0.1 ml of antivenomand incubated for 5 min at room temperature; then a0.1-ml portion containing 2, 4, 8, 11, or 24 mg of venomprotein, respectively, was injected intradermally intoguinea pigs. The development of the lesions wasobserved as a function of time by measuring twodiameters of the erythematous lesion at 30-min inter-vals for 2 h and comparing this to a 0.1-ml salinecontrol injection.The backs of two white guinea pigs weighing

approximately 600 g were shaved, and each animalreceived two intradermal injections of 0.1 ml of salinecontaining an average of 25 jg of venom protein.Around one site, four intradermal injections of 0.1 mland, under the site, one subcutaneous injection of 0.2ml of antivenom were made immediately. The othersite received identical injections of normal rabbitserum.

In the systemic passive immunization experiments,two white guinea pigs of approximately 420 g receivedintraperitoneal injections of 15 ml of sterile antiserum18 h before the intradermal injection of 0.1 ml of 25 jigof venom protein.The effect of ethylenediaminetetraacetic acid

(EDTA) on whole venom was determined by prein-cubating 0.2 ml of venom in saline (114 jig of proteinper 0.2 ml) with 0.04 ml of 0.02 M EDTA for 15 min at37 C. A 0.12-ml portion of venom-EDTA solution wasinjected intradermally into guinea pigs and rabbits.Controls consisted of injections of untreated venom(57 jg of protein per 0.1 ml) or 0.02 M EDTA. Anothergroup of guinea pigs was treated by intradermal andsubcutaneous injections of 0.1 or 0.3 ml 0.05 MEDTA, respectively, around a site.

Guinea pigs weighing approximately 550 g weregiven three 0.1-ml intradermal injections consisting of(i) 37 jg of venom protein, (ii) a venom-heparinmixture (0.1 ml of 74 jig of venom protein waspreincubated with 0.1 ml of 166 jg of heparin for 15min at room temperature) or (iii) 167 jg of heparin.Two additional guinea pigs received two intradermalinjections of 0.1 ml of 31 jig of venom protein followedimmediately by four intradermal injections of 0.1 mleach containing 420 ug of heparin around one site.The untreated site received four 0.1-ml intradermalinjections of saline. This experiment was also re-peated except that the heparin and control injectionsfollowed the venom injection by 1 h.

Heparin was administered systematically beforeand after injection of venom into another set of twoguinea pigs. An intravenous injection of 0.5 ml con-taining 834 jg of heparin preceded by 1 h an intrader-mal injection of 35 jg of venom protein in 0.1 ml. A

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subcutaneous injection of 0.5 ml of 584 ,g of heparinwas given at the time of the venom injection, and 4 hlater an intraperitoneal injection of 0.35 ml of 584 /sgof heparin was also injected into each guinea pig. Thecontrol guinea pig received similar injections of saline.Venom toxicity studies. To determine the dura-

tion of venom activity in tissues, approximately 15 ,gof venom protein in 0.1 ml of saline was injected intoone site on the back of a guinea pig and 0.1 ml ofsaline was injected into a control site. After 24 h theguinea pig was sacrificed and the area of the lesionand control site were excised and suspended in 0.5 mlof 0.25 M sucrose in saline. These tissues were gentlyhomogenized with a hand homogenizer. The suspen-sions were centrifuged at 1,500 rpm for 10 min at 4 Cand the supernatant fluids were collected. Three0.1-ml injections were placed intradermally into an-other guinea pig; these consisted of (i) supernatantfluid from the venom area plus an equal volume ofantivenom, (ii) supernatant fluid from the venom

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area, and (iii) supernatant fluid from the salinecontrol area. This experiment was repeated with alesion that had been allowed to progress for 5 days.Two guinea pigs were sacrificed; 15 min after

death, 0.1 ml of 62 and 78 Ag of venom protein,respectively, were injected intradermally to deter-mine whether the venom contained enough spreadingand proteolytic activity to cause tissue damage innonliving tissue. A living guinea pig received 78 mg ofvenom protein in 0.1 ml given intradermally.

RESULTSThe injection of eluates from slices of the

acrylamide gel into guinea pig, skin revealedthat the dermonecrotic activity localized 15 to21 mm into the gel (Fig. 1), correspondinggenerally with a dense protein staining band atapproximately 20 mm into the gel. No othermaterial from the gel caused necrosis.

I,.A * $

10 15 20 25 30 35

S i c e N u m b e r sFIG. 1. Electrophoretic separation of brown recluse spider venom

location of the dermonecrotic activity.on polyacrylamide gel indicating the

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BROWN RECLUSE SPIDER VENOM

Figure 2 illustrates the elution profile of wholevenom from the Sephadex G-75 resin accordingto optical density readings of tubes taken at 280nm. Three major fractions are apparent. Thefirst eluted at the void volume, beginning attube 18. the second eluted between tubes 31 and40, and the third encompassed a broad foot(tubes 48 to 63) before a more absorptive zonebetween tubes 63 and 76. Hyaluronidase activ-ity was restricted to tubes 23 to 29, which arelocated in the trailing edge of the first absorp-tion peak. Dermonecrotic activity was detectedin the second major peak, and all other fractionswere devoid of this activity.Photographs of two immunodiffusion plates

are presented in Fig. 3. In the top photograph,starting in the lower left well and proceedingclockwise around the center well (antiserum),the wells contained Sephadex-fractionatedvenom tube 20 (from Fig. 2), whole venom,Sephadex-fractionated venom from tube 25,and whole venom, respectively. The immuno-diffusion plate in the lower photograph is simi-lar to the inmmunodiffusion plate in the upperphotograph except that the lower left wellcontained whole venom; the next three wells inclockwise order contained Sephadex-frac-tionated venom from tubes 34, 60, and 70,respectively.The results indicate that the hyaluronidase

fraction (tube 25 from Fig. 2) and the dermone-crotic factor (tube 23 from Fig. 2) induced theproduction of precipitating antibody. It is alsoapparent that there is more than one compo-nent in each of these two biologically activefractions and that material from the last majorpeak (tube 70 in Fig. 2) did not induce precipi-tating antibody.

Immunoelectrophoresis of whole venom re-vealed at least six precipitation bands, of which

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all but two migrated to the anode (Fig. 4).Esterase activity was demonstrated in precip-

itates formed by simple immunodiffusion andimmunoelectrophoresis. A positive reaction wasseen as a fine sharp red-violet band that is seenas a black line in the photograph (Fig. 4). Noprotease, lipase, catalase, acid phosphatase,alkaline phosphatase, nor amylase activity weredetected by immunohistochemical techniques,nor was lipid detected in the immunoprecipi-tates.

Figure 5 indicates that 8 jig of venom proteinin 0.1 ml was completely neutralized by anequal volume of antiserum. The antivenom wasovercome by greater quantities of venom whichwere then able to display typical tissue destruc-tion.Table 1 presents the results obtained by local

and systematic passive immunization of guineapigs. Test animals that were locally or systemi-cally immunized were totally protected againstthe development of a necrotic lesion. The ani-mals received 25 ,ug of venom, which caused thedeath of untreated animals approximately 48 hinto the experiment. Animals that were sys-temically protected with antivenom survivedfor more than 5 days and revealed only ery-thema about the injection site. Animals pro-tected by local injections of antivenom at onlyone of two venom sites were protected only atthe antivenom site and succumbed within 24 h,apparently to venom released from the un-protected site.There was no significant difference between

the effect of EDTA-treated venom and thewhole venom upon lesion size and progress inrabbits. In guinea pigs, there was a slightdecrease in intradermal necrosis as comparedwith untreated whole venom. The treatment ofguinea pigs with intradermal and subcutaneous

.HyaluronidaseActivity 100

90

80

70

rotic Activity 60

50

4101

30'

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AA 1

Tubes

FIG. 2. Elution pattern of recluse spider venom on Sephadex G-75 indicating the location of thedermonecrotic activity and hvaluronidase.

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FIG. 3. Immunodiffusion analysis of venom. Ineach case the center well contained antivenom. Begin-ning at the lower left and continuing clockwise: (top)Sephadex tube 20, whole venom, tube 22, wholevenom; (bottom) whole venom, tubes 32, 45, and 50.Tube numbers refer to Fig. 2.

injections of' EDTA immediately after venomhad no inhibiting eff'ect on the development of'alesion.The et'fect of 167 ,ug of heparin preincubated

with '37 Ag of whole venom protein is shown inTable 2. This concentration of' heparin com-

pletelv neutralized the dermonecrotic effect of'

the venom. When heparin was administered byintradermal and subcutaneous injections imme-diately after venom, no necrotic lesion devel-oped even after 24 h as in the preincubationexperiment. When a site injected with venomwas not treated until 1 h after the inoculation,no protective effect was noticed. If' heparin wasadministered systemicallv before and after theadministration of venom, there was a protectiveeffect on the lesion development and also on thesystemic effect of' the venom (Fig. 6).

After the intradermal transfer of 0.1 ml of' asolution prepared from a venom-injected orsaline-injected site on a guinea pig, only thevenom-derived fluid created a lesion. This ex-periment was repeated with a lesion that hadbeen allowed to progress for 5 days. but upontransfer, no subsequent lesion was formed.There was no sign of a lesion 24 h after the

intradermal inoculation of whole venom (62 and78 og) into guinea pigs 15 min after sacrificingthe animals. The control, living guinea pigdeveloped a very large lesion and died 40 h intothe experiment (Table 3).

DISCUSSION

The separation of' the protein components of'the venom of' L. reclusa by polyacrvlamide gelelectrophoresis and Sephadex G-75 gel filtrationafforded a means for analyzing partially puri-fied fractions for their biological activitv. Der-monecrotic activity was localized in the f'irstmajor protein component at the top of' thepolyacrvlamide gel which contained 10 or 11other areas containing stainable protein. Allother areas of' the gel were devoid of' necroticactivity. Fractionation of' the venom on Sepha-dex G-75 columns consistently produced threemajor fractions with def'inite absorbance at 280nm. This can be compared with the resultsobtained by Suarez et al. (20) with L. laetavenom, in which two fractions were noted. L.reclusa hyaluronidase was eluted from theSephadex column just after the void volumewell in advance of' the dermonecrotically activefraction. Dermonecrotic activitv recovered fromthe Sephadex column and polyacrylamide gel isexpressed simply as biological activity and notas specific activity per microgram of' protein dueto the low protein content of' many of' thef'ract ions.Ouchterlonv immunodiff'usion studies re-

vealed six, and possibly seven, immuno-precipitin bands. This is an increase over thefour bands reported by Smith and Micks (18)and is probably related to the use of' hyperim-mune antisera specif'ic tor L. reclusa venom.

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BROWN RECLUSE SPIDER VENOM

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FIG. 4. Immunoelectrophoretic analyses of venom. (Top) Stained for proteins; (bottom) stained for esteraseactivity .

IY9

0 30 60 90 120

Minutes

FIG. 5. Neutralization of venom-induced lesions in

guinea pigs by antivenom. Lesion area includes boththe necrotic and ervthematous zone. Numbers withinparentheses indicate the number of animals for thattest dose.

The hyperimmune antisera can also account forthe increased number of' immunoprecipitin

TABLE 1. Protection ofguinea pigs against L. reclusavenom by local or systemic passive immunization

At 4 h, area At 24 h, areaNo. of (mm2) of: (mm2) of:

Treatment animals/group Ery- Ne- Ery- Ne-

thema crosis thema crosis

None ...... 3 440 0 430 120Intradermal anti-venom... 3 60 0 50 0

Intraperitonealantivenom .... 2 195 0 198 0

bands detected by using immunoelectrophore-sis.Immunodiff'usion plates and immunoelectro-

phoresis slides were treated as histochemicalpreparations and stained for lipid and enzymes.Lipid could not be detected in the immuno-precipitates. Of the enzymes. only esteraseactivity was noted. The identif'ication of' en-zymes in venom is also under study by the morecommon biochemical assays.Experiments have shown that the dermone-

crotic activity of the venom was neutralized byincubation with ant ivenom before injecting sus-ceptible animals. Since passive immunization isused for immediate short-term protection justbefore or after an exposure to certain toxic

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TABLE 2. Protection of guinea pigs against L. reclusavenom by heparin

At 4 h, area At 24 h, areaNo. of (mm2) of: (mm2) of:

Treatment animals/group Ery- Ne- Ery- Ne-

thema crosis thema crosis

None ........... 3 402 0 480 180Heparina ....... 3 0 0 0 0

a167 Ag of heparin preincubated with 37 /tg ofvenom protein for 15 min before intradermal injec-tion.

400

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Yax

-lo (

Untreated Animal.dead at 48 hours)

Tneated Animal

I~~~~~~~~~~~~~~~~~~~W0 .5 1.0 1.5 2.0 2.5 3.0 3.5 24 48

TIME nhours)

FIG. 6. Reduction in size of uenom-induced lesionsby heparin.

TABLE 3. Effect of L. reclusa venom on vital andnonvital tissue

At 5 h, area of At 24 h, area of

Tissue Ery- Ne- Erv- Ne-

thema crosis thema crosis

Vital ............. 952 0 1400 72Nonvital ......... 0 0 0 0

antigens, this technique was applied to reclusevenom. Such protection against recluse venomcould be obtained in guinea pigs that werelocally immunized. Immunization (antiseruminjected intradermally around a venom site)immediately after the injection of venom pro-

tected the animal against dermal necrosis, butthe animals died within 48 h. These resultssuggest the possibility of' two toxic factors in thevenom, a dermal necrotic factor and a systemi-cally active lethal factor that was not neutral-ized by antivenom in this experiment. Guineapigs were both locally and systemically pro-

tected against the venom when they received an

intraperitoneal injection of antivenom 18 hbefore receiving venom. These results indicate

that the route and time of' antivenom adminis-tration is decisive in the demonstration of theprotective effect of antivenom against the localand systemic effects of the venom. When an-tivenom was administered locally 1 h after theinjection of venom, the local and systemiceffects of the venom were not reversed and theanimal developed a typical lesion and usuallydied within 48 h. These results also indicate thathuman bite victims could probably not receiveantivenom soon enough to prevent lesion forma-tion.

Passive transfer of material f'rom a primarylesion 24 h after its development produced asecond, smaller lesion. Antivenom preventedthis lesion development. This demonstratedthat some of the venom remained localized andactive for at least 24 h. Material transferredfrom 5-day-old lesions was inactive. These re-sults suggest that the chronicity of' L. reclusalesions may be due to the stimulation of anautodestructive reaction in the host by somevenom component rather than the continuedactivity of some toxic component in the venom.

Neutralization of venom necrotoxin by otherthan antiserum treatment was examined withthe objective that an effective immunization ortreatment for the spider bite might be discov-ered. There have been numerous reports in theliterature (5, 8, 10) that EDTA inhibited proteo-lytic snake venom enzymes, thereby suggestingan essential role of' metals in the activities ofvenom toxins. Previous experiments in ourlaboratory (23) demonstrated that reclusevenom esterase activities were inhibited in vitroby EDTA; therefore, studies were undertaken todetermine the in vivo eff'ect of' EDTA on reclusevenom. The results obtained in this studycontrast with the results obtained by the treat-ment of snake venoms with EDTA since thechelating agent did not decrease the venom'stoxicity.The discovery that heparin has a neutralizing

effect on whole venom is in harmony with thework of Higginbotham et al. (11, 12) on snakeand bee venom. The neutralizing effects ofheparin on recluse venom were observed whenwhole venom was preincubated with heparinand when heparin was administered systemi-cally before venom. The systemic administra-tion of' heparin prevents venous thrombosis inhuman patients (2, 9). This might indicate twomechanisms of heparin action on recluse venomtoxicity: the neutralization of' venom toxins.and the inhibition of' the clotting mechanism.This second activity would prevent micro-thrombic aggregates of leukocytes and plate-lets from plugging the arterioles and venules at

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the venom site, which could be expected to re-

sult in local necrosis.Two enzymatic activities have been identi-

fied in recluse venom that may degrade biologi-cal structures found in mammalian tissues (23).It seems possible that the hyaluronidase hydro-lyzes the polysaccharide matrix of the epider-mis and that the esterase attacks protein or

lipoprotein components of this tissue. Experi-ments designed to determine whether venom

components caused necrosis directly or whethernecrosis and lesion formation were due to an

interaction between body constituents and thevenom revealed that no lesion developed whenwhole venom was injected into nonvital tissue.It is thus concluded that an interaction of bodyconstituents and the venom is essential for thedevelopment of a lesion. This is in agreementwith the data of Smith and Micks (19), whodemonstrated a need for complement and poly-morphonuclear cells for the development oflesions after injections of brown recluse spidervenom.

ACKNOWLEDGMENT

This research was supported in part by Public HealthService research grant ES00598 from the Division of Environ-mental Health Services.

LITERATURE CITED

1. Atkins, J. A., C. W. Wingo, W. A. Sodeman, and J. E.Flynn. 1958. Necrotic arachnidism. Amer. J. Trop.Med. Hyg. 7:165-184.

2. Bonnar, J., K. W. E. Denson, and R. Biggs. 1972.Subcutaneous heparin and prevention of thrombosis.Lancet 2:539-540.

3. Clausen, J. 1969. Immunochemical techniques for theidentification and estimation of macromolecules, p.529-536. In T. S. Work and W. Work (ed.), Laboratorytechniques in biochemistry and molecular biology.American Elsevier Publishing Co., Inc., New York.

4. Denny, W. F., C. J. Dillaha, and P. N. Morgan. 1964.Hemotoxic effect of Loxosceles reclusus venom: in vivoand in vitro studies. J. Lab. Clin. Med. 64:291-298.

5. Deutsch, H. F., and C. R. Dinis. 1955. Some proteolyticactivities of snake venoms. J. Biol. Chem. 216:17-26.

6. Dillaha, C. J., G. T. Jansen, W. M. Honeycutt, and C. R.Hayden. 1964. North American loxoscelism, necroticbite of the brown recluse spider. J. Amer. Med. Ass.188:33-36.

7. Duffey, P. H., and H. P. Limbacher. 1971. Brown spider

bites in Arizona. Arizona Med. 28:89-95.8. Friederich, C., and A. T. Tu. 1971. Role of metals in

snake venoms for hemorrhagic, esterase and proteolyticactivities. Biochem. Pharmacol. 20:1549-1556.

9. Gallus, A. S., J. Hirsch, R. J. Tuttle, R. Trebilcock, S. E.O'Brien, J. J. Carroll, J. H. Minden, and S. M.Hudecki. 1973. Small subcutaneous doses of heparin inprevention of venous thrombosis. N. Engl. J. Med.288:545-551.

10. Goucher, C. R., and H. H. Flowers. 1964. The chemicalmodification of necrogenic and proteolytic activities ofvenom and the use of EDTA to produce Agkistrodonpiscivorus venom toxoid. Toxicon 2:139-147.

11. Higginbotham, R. D., and J. M. Clark. 1967. Significanceof the local tissue responses to venoms in normal andsensitized mice, p. 337-349. In R. E. Russell and P. R.Saunders (ed), Animal toxins. Pergamon Press, NewYork.

12. Higginbotham, R. D., and S. Karnella. 1970. The signifi-cance of the mast cell response to bee venom. J.Immunol. 106:233-240.

13. Lewis, M. I., and J. F. Regan. 1966. Necrotic arachnid-ism. Calif. Med. 105:457-462.

14. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

15. Nance, W. E. 1961. Hemolytic anemia of necrotic arach-nidism. Amer. J. Med. 31:801-807.

16. Pearse, A. G. 1968. Histochemistry: theoretical andapplied, 3rd ed. Little, Brown and Co., Boston.

17. Pascale, J., S. Avrameas, and J. Uriel. 1966. The charac-terization of rat pancreatic zymogens and their activeforms by gel diffusion techniques. J. Biol. Chem.241:3023-3027.

18. Smith, C. W., and D. W. Micks. 1968. A comparativestudy of the venom and other components of threespecies of Loxosceles. Amer. J. Trop. Med. Hyg.17:651-656.

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20. Suarez, G., U. Biggemann, and H. Schenone. 1971.Biochemical studies of the venom of the spider Loxos-celes laeta and its mechanism of action. Bol. ChilenoParasitol. 26:60-62.

21. Uriel, J. 1963. Characterization of enzymes in specificimmunoprecipitates. Ann. N.Y. Acad. Sci.103:956-979.

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23. Wright, R. P., K. D. Elgert, B. J. Campbell, and J. T.Barrett. 1973. Hyaluronidase and esterase activities ofthe venom of the poisonous brown recluse spider. Arch.Biochem. Biophys. 159:415-426.

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