Determining which Rattlesnake Have Hemorraghic

Toxins and Which Have Mojave Toxin.

Jullieta Ornelas, Gilbert Limon, Dr. Eppie Rael

National Science Foundation Bridges to the Future

University of Texas at El Paso and El Paso Community College

 

Introduction

            The main objective of the work I performed this summer was to determine which rattlesnake venom contained either hemorraghic toxin, Mojave toxin, or both. According to Dr. Leib “There are thirty different rattlesnakes found in the world. In North America the number found is sixteen, and in and around El Paso County we find 6.”

            Hemorraghic toxin causes hemorrhaging. It is one of numerous enzymes found in rattlesnake venom.  It is a proteolytic enzyme which is considered to be metalloproteases and needs Zn2+ (Zinc) to function. This zinc is removable with a chealating agent such as with E.D.T.A..

            Metalloproteases cause the hemorrhaging itself.  Metalloproteases are fibrinolytic, which means that they breakdown fiber in clots.  They are also fibrinogenolytic, which means that they breakdown fibrogen and may cause the formation of blood clots.

            Mojave toxin is a neurotoxin.  Neurotoxins prevent neurotransmission, causing paralysis.  Mojave toxin is a neurotoxin with Phospholipase A2 activity.  It is also one of numerous enzymes that are found in rattlesnake venom.

Methods and Materials

            The collection of the venom was done by allowing the snakes to bite a parafilm-covered beaker.  These venoms were then frozen and lyophilized.  They were identified by labeling the container with information such as the type of snake found, a example is CSS 19-1 which indicates a Crotalus scrutulatus scrutulatus (Mojave Rattlesnake), and also the first time that the venom was collected and the location where the snake was collected. 

            Isoelectric focusing was used to separate the proteins in the venom based on the isoelectric charge of the protein.  First an agrose isogel was prepared and placed on the electrophoresis apparatus.  Next a dilution of 10 mg of powdered venom to 1 ml with d-water (stock solution) was placed on a preparation mask on the gel to make a fixed gel. The dilution for a transblotted gel was 1/10 dilution of the stock solution.  Catholytes and anolytes were used on opposite sides of the apparatus in order to conduct an electrical current with which to separate the protein in the venom. Separation of the proteins was done with a ten minute pre-focus time followed by a thirty-five minute timed increment of electrophoresis. The  gels were then either fixed or transblotted.

            Fixed gels were placed first in a fixative solution for two minutes undisturbed then ten minutes agitated.  After this step the gels are be cleansed in d-water and dried in order to stain them. Staining was done with a comassie blue solution.

            A transblot was prepared with a transfer membrane prepared in 70 % MEOH in order for it to absorb the venom. The gel was then placed on scoth brite pads with the membrane on it.  This was then placed inside of the transblot cell box which contained 17.5 ml of acetic acid and 2.5 liters of d-water and left running for one hour. After this we would incubate the membrane was incubated for 15 minutes in blotto.  The membrane was washed, then the primary antibody was added along with 10 ml blotto and left incubating for two hours. After the two hours, the membrane was washed and incubated for two more hours with the secondary antibody and 10 ml blotto.  Finally the membrane was once again washed with d-water and developed with HRP. The HRP was made with 8 ml d-water, .9 ml buffer, .1 ml hydrogen peroxide (3%), and 1 ml 4chloro2naphthol.

            Venoms used in this experiment included those of Crotalus molossus molossus (CMM Northern blacktailed Rattlesnake), Crotalus scrutulatus scrutulatus (CSS Mojave Rattlesnake), and Crotalus viridis viridis (CVV Praire Rattlesnake).

            The primary antibodies used were CSS-12 which recognizes Mojave Toxin, AF5 which recognizes metalloproteases, and CAP-8 which recognizes metalloproteases including Hemorraghic toxins. Also monoclonal antibody XX was used which recognizes Mojave toxin.

            The importance of this lies with the posibility of surveying within and across the rattlesnake species.  Physicians should be aware that venoms are not all identical. Also the systematic classification of the venoms could be useful for futher refrence. A practical use of enzymes found in rattlesnake toxin is by using the metelloproteases to produce an anticlotting agent that may be used for preventing heart attacks by eliminating blood clotts. Also the opposite could be benificial,  by using the enzymes to produce a blood clott in blood vessels leading to tumorswe could essentially eliminate the tumor. If this type of medicine is to be useful, we would need to determine which snakes have which venoms for when we need to use them.

Results and Discussion

             Of nine diffrent Crotalus scrualatus scrutalatus venoms tested for hemorraghic toxins with the CAP-8, only came up with a positive reaction which was CSS 36-9. CSS36-9 also came up possitive with XX and AF5 (test which were conducted by Julieta Ornellas). This means that CSS 39-9 was possitive for both hemmoraghic toxin as well as Mojave toxin. According to the XX tested membrane (Julietta Ornellas), CSS was found to test possitive more for Mojave toxin.

            Also tested were CVV 19-1, CVV? DIL 236, CMM TJL 340, and CVV14-1. Although results were not clear, my hypothesis is that the Crolatus viridis viridis samples contain a stronger hemorraghic toxin result than the CMM samples, based on a test done on July 1, 98 where results came up possitive but not where they were expected to be found on the membrane.

            Also because of a technical problem, later found to be the fixative, results for the same experiment were negative when they were tried to be duplicated.

Acknowledgements

Reasearch and funding for the Bridges Program was funded by the National Science Foundation.

Literature Cited

1.  Aird S. D., Thirkhill L. J., Seebart C. S. and Kaiser I. J. (1989) Venoms and Morphology of western diamondback/Mojave rattlesnake hybrids. J. Herpetol. 23, 131-141.

2.  Klauber L. M. (1972) Rattlesnakes. Their Habits, Life Histories and Influence on Msnkind. 2nd edn. Universityof California Press, Berkeley, CA.

3.  Mayer M. M. (1967) Complement and complement fixation. In Experimental Immunochemistry, (Edited by Kabat E.A. and Mayer M.M.), p. 133. Thomas, Springfield, IL.

4.  Rael E.D. and Jones L.P. (1983) Isolation of an anticomplement factor from the venom of the Mojave rattlesnake (Crotalus scrutulatus scrutulatus) venoms and migration diffrences of Mojave toxin. Toxicon 21, 57-65.

5.  Rael E.D., Johnson J.D., Molina O. and McCrystal H.K. (1992) Distribution of a Mojave toxin-like protein in Crotalus lepidus (Rock rattlesnake), In Biology of Pitivipers (Edited by Cambell J.A. and Brodie E.D. Jr), pp. 163-168. Selva Press, Tyler, TX.

 

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