Odell, George

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OSU Journals (Oklahoma State University)

PHYSICAL SCIENCES 268Protein Components of Loxosceles reclusa VenomGEORGE ODELL, NAZEEBA NAZBAT, ROGER GROTHAUS' andD. E. BOWELL, Oklahoma State University,Departments of Biochemistry and Entomology, StillwaterThe availability of certain spider venom preparations through bio-logical supply houses, such as Sigma Chemical Co. (1966), and the recog-nition that spiders other than Lathrodectw mactana create a medical prob-lem indicate that more infonnation is needed concerning spider venomcomponents. A recent report on arachnidism in Texas by Micka (1963 )revealed that Lox08ce"le8 reclU8a~ the brown or fiddleback spider, is agrowing problem for this region. Studies on the characteristics of venomfrom this spider by Denny and co-workers (1964) showed that it wasstable when freeze-dried or in suspension, was inactivated by heating at56 C for 10 min, and was active as a red blood cell hemolytic agent. Theseworkers also report the appearance of antibodies "to some portion of thecrude venom extract . . . by immunodiffusion studies."From the venom characteristics reported by Denny (1964) and thenature of the cytotoxic reaction, enzymatic actiVity tor the active com-ponent of this venom was suspected. Separation of the protein compo-nents of L. reclU8a venom was accomplished by an electrophoretic tech-nique which utilized the polyacrylamide gel disc apparatus. Protein com-ponents obtained by this method were checked for biological activity andfurther studies on the enzymatic actiVity of these components are in pro-gress.EXPERIMENTAL PRocmUREsThe development of a technique for recovery of pure spider venom(Grothaus and Howell, 1967) has provided material for examination byelectrophoretic separation methods. Since the spider L. reclusa yields ap-proximately 10 pg of venom per "milking," 10 spiders were used to pro-vide venom for one electrophoretic separation. Duplicate samples wererequired as it is necessary to stain one separated sample to mark theposition of protein components in the second sample. Twenty spiders werethus required for each experiment. The Conalco polyacrylamide gel discelectrophoresis technique was used with a glycine-tria butfer, pH 8.5. Thepaper pad used to collect the venom was inserted in the upper end of theelectrophoresis tube and gel suspended in butfer was poured around thepad. '!'he samples were subjected to electrophoresis for approximately35 min at 5 rna per tUbe. Voltage was from 100 to 250 v depending onthe number of tubes used in the apparatus. Atter electrophoresis, onetube was stored at 4 C while the other was stained and destained to showthe position of protein components. The unstained gel was sectioned toseparate individual protein components by using the stained gel as apattern. This system has the sensitivity to separate and detect the pro-tein components in 0.1 mg of pure venom.Resuspenslon of the protein was effected with 0.2 ml of normal saline.The gel and saline were miXed in the tip of a conical 15-ml centrifuge tUbe.This mixture was held at 4 C overnight; then low speed centrifugationgave about 0.1 m1 of clear supernatant tor blo-assay. Biological actiVityof the resuspended protein fraction was determined by injection of thisILt. IISC. USN, and &T&daate student, Oklahoma State Universlt)'. The opinionand ...ertioDS contained herein are the priva~ ones of the authors and are Dot to beCODatraed .. official or relJeetlq the views of the Nav)' Department or tbe Naval &erv-iee at1 q ..2M PROC. OF THE OKLA. ACAD. OF SCI. FOR 1966ruu.pended protein into an adult male American cockroach. A 5-hourobHrvatton time wu used as the most representative of toxic effects.The control (c) would represent extraction of a section of the samplegel atrip which shows no protein bands.RESULTSThe number and relative intensity of protein components in L. reclusavenom tor each sampling date are shown in Figure 1. The fraction num-ber in th18 figure i8 used in Table I to designate protein component.DISCUSSION OF RESULTSThe disc electrophoretic system described under methods has beenused to separate the major protein components of pure L. reclusa venom.Five to seven bands or protein components were observed using this sys-tem with 0.1 mg ot venom. Two to three of these protein componentswere found to have biological activity when saline suspensions were in-jected Into cockroaches. Protein fractions designated 3, 4 and 5 showthe greatest biological activity based on the roach assay. Further workin the characterization of these biologically active components as hemo-lytic agents and as specific enzymes is in progress. Variation in thenumber of bands and position is being studied with reference to electro-phoretic technique and date of venom collection.9/8/66dye 12 3 4 56(......-IIIIllIJII-----..."OC 12 34 5 6 7C 123456 79/22/6610/4/66c c 234 511/15/66I1aure 1. Polyacrylamide gel electrophoretic separation of L. recl.aovenom (0.10 mg)PHYSICAL SCIENCES 266TABLE I. TOXICITY· OF PRoTEIN COMPONENTS OF L. recu18a VENOMCondition of Roach 5 Hours After Administration of VenomProtein ComponentFraction 9/8/66 9/22/66 10/4:/66 11/15/66Control ..........._._._........ No change No changet No changeControl .....-.........._........ No change No change No change1 No change No change No change No change2 No change No change No change No change3 Dead No change No change No change4 Dead On back No change Dead5 No change On back No change On back6 No change No change No change7 ........................... No change No change------------------------- _._------_.- ----_....*Resuspended protein band equivalent to 0.05 mg venom injected intoAmerican cockroaches.t Venom protein concentration very low.ACKNOWLEDGMENTThe technical assistance of Mrs. Tova Auda in the electrophoreticprocedures used in this work is gratefully acknowledged.LITERATURE CITEDDenny, W. F., C. J. Dillaha and P. N. Morgan. 1964. Hemotoxic effectof Loxosceles reClu,SU8 venom. In vivo and in vitro studies. J. Lab.Clin. Moo. 64 :291-8.Grothaus, R. H. and D. E. Howell. 1967. A new technique for the recov-ery of spider venom. J. Kans. Entomol. Soc. 40(1) :37-41.Micka, D. W. 1963. The current status ot necrotic arachnidism in Texas,in Venomous and Poisonous Annna18 and Noxious Plants 01 the Pacificregion. The Macmillan Co., New York, p. 153.Sigma Chemical Co. Catalogue. 1966. Venoms, p. 194.

Protein Components ofÂ Loxosceles reclusaÂ Venom

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Protein Components ofÂ Loxosceles reclusaÂ Venom

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