Presented by Grace P. Goracci Class of 1979 SWEET BRIAR COLLEGE LIBRARY Sti'CET E!!!^-- CCllEGE IIPRARY SlvirT t.MAA. VA 2<595 Digitized by the Internet Archive in 2010 with funding from Lyrasis Members and Sloan Foundation http://www.archive.org/details/studyofvenomofglOOgora A STUDY OF THE VENOM OF GLYCERA. DIBRANCHIATA EHLEftS by Grace P, Goracci ■."7li^ji,if71 Date Approved: Ml. L^U^^J^ Thee^s^dviser /jDutside Reader A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree with Honors in Biology-Chemistry Sweet Briar College Sweet Briar, Virginia April, 1979 TABLE OF CONTENTS INTRODUCTION 1 Background 1 Morphology of the Proboscis 1 Venom Analysis 6 MATERIALS AND METHODS 8 Dissection Methods 9 Preparation of Crude Extracts H Methods of Extract Analysis 1/f Toxicity Assay lilf Acetone Assay 15 Trypsin Assay 15 Folin-Ciocalteu Assay 16 Gel Permeation Chromatography 1? Sephadex G-lOO 1? Sephacryl S-200 17 RESULTS AND DISCUSSION ZZ Preliminary Studies on Crude Extracts 2.Z Chromatography 30 Sephadex G-100 30 Sephacryl S-200 32 CONCLUSIONS ^1 SUMMARY k3 LITERATURE CITED kh O l'7SeS7 ACKNOWLEDGEMENTS I would like to thank Dr. Margaret Simpson and Dr. Helen Gager for their advice and guidance during this study, I would also like to thank Mr. Vi/arren Jones and Dr. Barbara Blair for their advice. The photographs appearing on pages 10-13 were done by Catherine Harold. Gratitude is extended to Catherine, as well as to Carmen Maegli, who helped with some of the chromatographic standards. O r STATEMENT OF PURPOSE The goal of this study is to separate the toxic principles of the venom of Glycera dibrajichiata. of which little is known, and to compare these components to those partially identified in Glycera convoluta(Michel 1966,1972,1975). Gel chromatography, being a well established technique for separating large molecules according to size, is the primary separation method chosen for this study. Stability of the crude extracts as a function of temperature will be determined before the separation. This study is expected to reveal biological and chemical information concerning the poison glands of Glycera dibranchiata^ which could prove to be of biomedical importance. t) ( INTRODUCTION Background Glycera dibranchiata . commonly known as the bloodworm, is a marine annelid of the family Glyceridae, It was first described by Ehlers in 1868 ^nd is an errant polychaete. The common name refers to the reddish color of the worm which is due to the hemo- globin found in the coelcmic cells(Klawe & Dickie 1957). The bloodworm inhabits subtidal zones from northeast Canada to North Carolina (Klawe & Dickie 1957). The anatomy of the bloodworm has been described in detail by Ehlers (1868) and Klawe and Dickie (1957), and in less detail by Halstead(1959) and Heacox(197A-). Glycera dibranchiata has been known to reach a length of 37 cm, but the average length is about ^ 25 cm(Klawe & Dickie 1957). The worm has a long cylindrical body which is tapered at both ends. At the anterior end of the body the bloodworm has an eversible pharynx. This first third of the digestive tract can be rapidly protruded, and has four sharp, black, chitinous Jaws at its anteriormost point(Fig 1), Four glands, first referred to as poison glands by Ehlers (1868), are each separately associated with a jaw by a connecting duct. It is believed that the proboscis, and its associated poison-gland complex, is used for procurement of food and burrowing purposes. Morphology of the Proboscis Heacox(197/f), in a histological study of the poison-gland complex, refers to three parts of the proboscis: an eversible Aj buccal tube, a pharynx with jaws and associated glands, and an esophagus ue to the size of the glands it was not possible to separate them from these muscle layers. Therefore, esophageal muscle from each worm was used as a controKFig 12), 10 Fig 5, Jaws of Glycera dibranchiata can be seen through the body wall. Fig 6. Longitudinal cut from just posterior of jaws toward anterior end of bloodworm. < 11 Fig 7, Looping proboscis over scissors, BT-buccal tube, J- jaws, G-glands, E-esophagus. Fig 8. Holding the esophagus between the index finger and thumb. BT-buccal tube, J- jaws, G-glands. 12 Fig 9. Holding the buccal tube between the index and middle fingers, BT-buccal tube, J- Jaws, G- glands, E-esophagus. Fig 10. Dissection a gland, J- Jaw, G-gland, BT-buccal tube. 13 Fig 11, First third of digestive tract with glands removed, BT-buccal tube, J- Jaws, Ph- pharynx, E-esophagus, Fig 12, Dissecting esophageal muscle for control, Ph- phary nx, E-esopha gus , c 1^ Preparation of Crude Extracts All equipment which was in contact with the tissues for any length of time was kept on ice throughout the procedure to help prevent histolysis. The dissected poison glands and muscle controls were placed in separate small, plastic weighing trays during the dissection. The trays contained a few drops of sea water to prevent the tissue from drying out. Each sample was then triturated in a homogenizer or, if more than 15 worms per sample was used, the tissue was ground in a mortar and pestle with clean sand. Depending on the sample size, up to 1.0 ml of sea water was added throughout this process. The samples were centrifuged in a Beckman TJ-6 at 3000 RPM, 6°C, for 10 min. The extract usea on the Sephadex and Sephacryl columns consisted of ^00 glands in k.O ml of 5raM NH, HCO,, centrifuged at 5800 RBI, 6°C, for 2.5 hrs. Method of Extract Analysis Toxicity Assay The toxicity of the extracts was determined by injecting Uca with a small amount of crude extract. These animals are hardy and readily obtainable, and it had previously been established by Curtis(197^, unpublished) that the venom was highly toxic to them. The 'standard' dose was determined in the following manner: first, the total weight of the tissue (gland or muscle) and sea water was recorded. From the measured volume and specific gravity of sea water, the weight of the tissue &lone was determined. The density of the extracts was calculated in terras of grams of 15 tissue per ml sea water. The 'standard' was set at 6.0 mg W tissue per unit(l,70 gm) of crab weight. Injection of this standard dose caused complete cessation of movement in a crab within 5-10 min. The stability of the venom extract was determined in terms of the standard Tab toxicity. Extracts were subjected to the following conditions: -20°C up to 35 days, 22°C up to 2.5 hrs, and 100°C for 10 rain. The muscle control extract was tested under the same conditions. The extracts were found to be stable for at least 35 days at -20°C. This form of storage was used throughout this study. Acetone Assay The gland and muscle extracts were first tested for the ^ presence of protein. This was done by adding varying amount of acetone to the extracts (Clark & Switzer 1977); the ratios of acetone to extract were approximately 0.5^1 » l-l, and 2:1, After each addition of acetone, the extracts were thoroughly mixed and then centrifuged at 2000 RPM, 6°C, for 10 min. The super- natant was poured off and re-extracted with acetone to obtain the next ratio. The precipitates were redissolved in sea water and tested for crab toxicity. Trypsin Assay Both crude extracts were tested for trypsin activity accord- ing to the procedure of Schwert and Takenaka(1955). Trypsin activity was measured in terms of the change in spectrophoto- metric absorbance at 253 nm and 20°C over time; the absorbance 16 was a measure of the amount of N-«^-benzoyl-L-arginine ethyl ester(BAEE) hydrolyzed by the trypsin. The solutions needed for this assay were O.OOIM BAEE in 0,05M tris-hydroxyme thy laminome thane and a trypsin solution in O.OOIM HCl. The absorbance was set at zero with O.OOIM BAEE in 0.05 tris buffei in the reference cell. 0.2 ml of the trypsin solution was then added to 3.0 ml of this BAEE solution in the sample cuvette. The optical density initially decreased due to the Qiiution of the BAEE by the trypsin solution. However, the absorbance then increased linearly until about 90% of the substrate was used(Rick 1965). The crude extracts were tested at varying amounts, up to 0,50 ml, Folin-Ciocalteu Assay Protein concentration of the extracts were determined according to the Folin-Ciocalteu assay. The reagent yields a bluish color when mixed with free or protein-bound tyrosine and try ptophaji( Clark & Switzer 1977). The protein concentration was measured spec tro photometrically , The presence of these two amino acids is a good measure of protein concentration since their total quantity in most soluble proteins is constant. This assay is very sensitive and is quantitatively accurate up to 300 xLg protein. The protein standards consisted of lysozyme in the follow- ing concentrations: 50, 100, 200 and 300 Ag, Each standard solution had a final volume of 1 .2 ml. A blank, consisting of 1 .2 ml of HpO, was also used. An alkaline copper reagent was freshly prepared by mixing the following solutions in order: « 17 1.0 ml of 1% CuSO 'H 0, 1.0 ml of 2% sodium tartrate and 98.0 ml of 2.% Na^CO in 0.1N NaOH, To each standard and the blank, 6.0 ml of the alkaline copper reagent was added. After 10 min, 0.3 ml of Folin-Ciocalteu reagent was mixed in. The absorbance of each standard was read at 500 nm against the blank after 30 additional rain, A range of crude extract concentrations was tested, up to 0,20 ml. Gel Permeation Chromatography Sephadex G-lOO Gel permeation chromatography was used to separate the protein components of the venom extract. The column was a Pharmacia K16/70, 1,6 X 70 cm, including a reservoir and a 3-way valve (Fig 13). The crude venom extract used for the column, prepared similarly to the Glycera convoluta venom extract used by Michel (1975), consisted of i+OO glajids in 4.0 ml of 5mM NH. HCO,, About 1.0 ml of this extract was put on the Sephadex column and run at 6°C, Fractions of 3.0 ml were collected at a flow rate of 0,12 ml/min, which slowed down considerably as the sample passed through the column. After collecting about 67 ml the column was stopped. The fractions were monitored for protein content by spectro photometric absorption at 280 nm. Various fractions were tested using the previously described toxicity assay, Folin- Ciocalteu assay and trypsin assay, Sephacryl S-200 The same column, K16/70, was used as for the Sephadex G-lOO (Fig 13). The column was packed as recommended by Pharmacia, ( 18 Fig 13. Pharmacia K16/70 column used for Sephadex G-100 and Sephacryl S-200 separations. 3-way valve adaptor 19 Fig 1/f. Pharmacia K16/70 column being packed with reservoir directly attached to column. tije»tODtf> r 20 Approximately 200 ml of Sephacryl S-200 Superfine was used, which is about 125% of the packed bed volume. The gel was suspended in eluant buffer, 5mM NH, HCO,. The amount of buffer used for the suspension was about three times the volume of the settled gel. After the gel resettled, the excess buffer was decanted off. Then enough buffer was added to make a slurry of 70% settled gel and 30% excess liquid. The slurry was poured into the column and allowed to settle. The reservoir, connected directly to the top of the column(Fig 14), was filled with eluant buffer. A second reservoir was positioned above the column to obtain a flow rate of about i+O ml/cm -hr. The column was eluted with buffer for two hours. The reservoir attached direct- ly to the column was removed, and a flow adaptor was added, so that it was just touching the gel surface(Fig 13). The direction of the flow was reversed and the column was eluted with the buffer at the same rate, for two hours. Upon completion of the back flushing, the column was packed and ready for chromatography at a flow rate of 30 ml/cm -hr or less. The packed bed volume was 12/+, 6 cm-^, A sample of the same batch of venom extract that was used on the Sephadex G-100 column was used here. The sample consisted of 1,5 ml extract, which was eluted with 5mM NH, HCO;, buffer. Fractions of 3.0 ml were collected at a flow rate of about 1,0 ml/min. The protein content was again monitored by recording the spectro photometric absorbance at 280 nm. The toxicity aissay, trypsin assay, and the Folin- Ciocalteu assay were performed on selected fractions. The Sephacryl column was calibrated by using Pharmacia high and low protein molecular weight calibration kits. Table 1 21 Table 1 , Proteins used in Calibrating Sephacryl S-200 Column, Protein Volume on column(ml) mg protein per ml buffer r Blue Dextran 2000 1.0 1.0 II Thyroglobulin 1.0 2.0 III Thyroglobulin Catalase 1.0 5.0 5.0 IV Aldolase Ribonuclease A 1.5 5.0 5.0 V Thyroglobulin Aldolase Ovalbumin 1.0 2.0 2.0 7.0 VI Thyroglobulin Albumin Ribonuclease A 1.0 2.0 7.0 7.0 shows the proteins that were used. Except for the Blue Dextran 2000, the proteins were eluted with the buffer at pH=7.8. When the Blue Dextran was run at this pH, it was absorbed by the gel, Therefore, the pH of the buffer was increased to about 10 by addition of NaOH, and the Blue Dextran was successfully rerun. 2Z RESULTS AND DISCUSSION * Preliminary Studies on Crude Extracts All Uca pugjlator injected with a standard dose of venom extract showed the reactions of envenomation as listed in Table 2, Except for the initial scurrying, which was shown by both experi- mentals and contiols, these symptoms are the signs of venom toxicity. The control crabs scurried around immediately after the injection, and then appeared normal. The results of the toxicity assay are summarized in Table 3. In order to test for stability, extracts were stored at -20 G for up to 35 days. When the crabs were injected with the venom extracts (Table 3, sample #4), they exhibited the same toxic symptoms as recorded in Table 2. The control crabs did not show any unusual signs. Therefore, the extracts may be considered stable, for the purposes of this experiment, at -20°C for 35 days. The results of the study at room temperature suggest two active principles in the venom, a quivering factor and a lethal factor. The latter showed no change when the extract was stored at 21-22°C for up to 2,3 hrs; however, the quivering factor de- creased in activity under the same conditions. The injected crabs exhibited less quivering the longer the venom remained at room temperature, and after 2.5 hrs no signs of quivering were observed. Therefore, this quivering factor appears to be a temperature-sensitive protein which has an activity inversely proportional to the length of storage time at room temperature. Heat stability of the toxin was tested by heating the ^ extract at 100°C for 10 min, A large amount of coagulum appeared, indicating the presence of proteins. Then the extract was 23 Table 2. Signs of envenomation. Time 10-15 sec 30 sec 50-90 sec within 20 sec 2-/+ min 5-10 min Observation immediate scurrying legs on injected side of body start quivering legs on injected side straighten and become limp eyestalk on injected side falls other eyestalk falls crabs generally tilt backwards - there are alternate periods of legs quivering, legs appearing limp, and then legs straightening out smd quivering - no reaction made when threatened no movement ^ Table 3, Results of toxicity assays, 24 Number Sample crabs/ number test Treatment of extracts Cessation of crab movement Expt's(min) Cntrl(days) 1 7 Stc.r.d£rd 5-10 2,3,3,3,4, 21,31 la 2 Sea-water control - 4.32 Frozen(-20°C) 2 2 few days 5-10 - 3 2 5 days 5-10 ^.s k 2 35 days 5-10 14,40 Heated (100°C, 10 min) 5 2 cooled slowly 3,^ days 39,100 6 2 cooled slowly, centrifuged 5,8 days 5,6 7 1 cooled immed,, centrifuged k days — 8 2 Room temperature (21 -22°C) 15 min 5-10 30 min 3 days 9 2 2^ min 5-10 1,3 10 2 /+5 min: little quivering, 5-10 1 hr, 4 day 11 2 1 hr: less quivering, 5-10 2,43 12 2 1,5 hr: less quivering. 5-10 3,43 13 2 2 hr: less quivering, 5-10 2,3 U 2 2.5 hr: no quivering. 5-10 •* Acetone precipitation 15 2 fraction #1 35 4,4 16 2 fraction #2 3 4,4 17 2 fraction #3 6 4,4 -not tested * see text 25 allowed to cool to room temperature (samples 5,6) and injected ^ into the crabs. The crabs were very lethargic for 10-15 min after the injection, showing only slight reactions. Initially it was thought that some of the coagulate had been injected into the crabs, and that this could somehow be causing the lethargic symptoms (sample "^O. The test was repeated with another sample (6), centrifuging the heated extract(after it cooled to room temper- ature) at 2500 RH4, 5°C, for 20 rain, so as to eliminate that possibility. However, these injected crabs showed similar behavior. A third possible explanation is that some of the protein could be re naturing(Freif elder 1976) since the extract had been allowed to cool slowly at room temperature. Therefore another venom extract was pre pared (sample 7); it was similarly heated for 10 min, immediately cooled in an ice bath, and centrifuged * at 2500 RPM, /f°C, for 10 min. These crabs showed no signs of venom toxicity; thereby supporting the third explanation. The concept of renaturation seems to be a recent topic. Much of the terminology used is only vaguely defined, A 'de- natured' enzyme refers to "a form of a macromolecule that has less secondary structure than that which is called native" (Freifelder 1976). The 'native' structure is defined as being the macro- molecular structure as found in nature, the isolated but enzyraatically active structure of a macromolecule, or that structure which retains its secondary spatial arrangement, but is biologically inactive (Freifelder 1976), The kinetics of denaturing enzymes is very complicated (Roberts 1977). The process depends on the number of bonds altered, and is associated with a high enthalpy of activation 26 and a large increase in entropy (Roberts 1977). The entropy change I suggests that the protein loses its ordered structure, including the active site. Most enzymes denature at 70°C, although exceptions have been found(Roberts 1977). Of the enzymes studied so far, trypsin, which has been identified in bloodworm venom, has a high degree ci renaturation(Neilands I964). Smaller proteins are able to almost completely renature if they are subjected to only mild methods of denaturation(Haurowitz 1963), The methods used in this study are considered only moderately harsh, which could account for a high degree of renaturation. In a study of octopus toxin, Ghiretti(1960) found that it produces the following effects: When a drop of saliva is injected into a crab, the legs immediately contract and the ajiimal remains in this position for about 1 min, f The righting reaction is abolished, the appendages begin to tremble, the pincers open ajid close without external stimulation, and tetanic spasms of the body at intervals are seen. Little by little the animal be- comes quiet; spontaneous movements as well as provoked reactions disappear and the aggressive animal of a few minutes before is changed to an inert limp orgauiism, , ,, The saliva has produced first a phase of overexcitability followed by a quiet phase and then paralysis, (p 730) These reactions are similar to those produced in this study by injecting crabs with bloodworm venom. Several of the components identified in octopus saliva are referred to as being very heat resistant(Ghiretti I96O); they are tyramine, histamine, acetyl- choline, taurine, p-hydroxyphenyl-ethajiol-amine(octopamine) and 5-hydroxytryptamine(5-HT, serotonin, enteramine ), When saliva was heated for 10 min at 100°C and injected, no lethal effects were observed, as was found in this study in the case of the 27 immediately cooled bloodworm venom. However, if there are heat # resistant substances in the bloodworm venom, as in the octopus saliva, it could explain the sluggishness of the crabs ofter being injected with heated and slowly cooled venom, Ghiretti did not say how the octopus saliva was cooled, but it would be interesting to s*-^ the effects of the different treatments on crabs. Because the appearance of a heavy coagulum in heated extracts suggested the presence of proteins, acetone precipitation was used to fractionate, or partially purify, the extracts. Pre- cipitates were obtained by adding different amounts of acetone to extracts, with the least soluble proteins precipitating out firs t( Clark & Switzer ^^Gk)^ After the first acetone extraction, a much larger amount of precipitate was noticed in the experi- mental sample than in the control. The opposite was true for the second and third extractions. When the venom precipitate was redissolved in sea water and injected into the crab, it became evident that a separation had taken place. The first fraction caused signs of envenomation, but at a very slow rate. The crabs did not completely stop moving for 35 min. However, the second and third fractions caused the same symptoms to occur atarauch faster rate, 3 and 6 min respectively. Another interesting observation was that fraction 1 had a lot more pre- cipitate than fraction 2. yet the latter fraction caused a much faster reaction to occur in the crabs. The active components of the venom must be very potent. These results seem to indicate that the toxic principles are not very soluble, and that the spreading factor(which is believed to cause tissue histolysis; is 28 more soluble than the toxic components themselves. If there are f two such components, they appear to be complementary in action. One speedily breaks down the tissue to allow the other component to penetrate quickly. The crabs injected with control precipitate did not exhibit any venomous signs. Acetone extraction on octopus saliva shows different results (Ghiretti I960). When the precipitate is redissolved in sea water and injected, the crabs show only overexcitability, and no sluggishness. The crabs then appear normal after a few minutes. Acetone precipitation may offer a second means for storing the venom; however, further studies need to be done to test its stability in this form. There is also a possibility that not all of the toxic components are proteins. In general, none of the control crabs showed signs of en- -^ venomation. Most crabs continued moving for a week or two, although the actual extremes were from 30 min to 100 days. The venom was tested for trypsin activity, yielding positive results (Table /+), The control was found to have very little, if any, trypsin activity. The presence of trypsin in the venom could account for the high degree of renaturation found in the heated extract. The Folin-Ciocalteu assay measured protein concentration up to 300 ^g. The control extract had 3-A(. mg more tissue per ml than the venom extract, although the latter showed a higher protein concentration( Table 5), It was not possible to accurately determine the protein concentration in the venom extract because the assay is valid only up to 300 yUg and the venom extract had a higher spectro photometric reading than that of the 300 Mg ( 29 Table if, Trypsj.i determination of crude venom extract. trypsin abs(253nm) time(min) ml extract abs(2^^nm) time(min) 5.0 2,0 1.0 0.600 0.109 0.057 0.50 0.05 0.05 o.3ifO 0.318 0.177 Table 5. Protein concentration of crude extracts. protein abs 500nm ml venom extract abs 500nm ml muscle extract abs 500nm 50 0.1/+/+ 0.05 0. if if 5 0.05 0.285 100 0.2/f5 0.10 0.573 0.10 0,if82 200 0.302 0.20 1.283 0,20 0.619 300 0.3^6 30 standard. These results do indicate that there is a higher f protein content in the venom extract than the control. Chroma to gra phy Having established the presence of proteins in the venom, the next step wac to try and isolate them. Gel permeation chroma- tography was chosen because it is a basic separation technique, Sephadex G-100 wa^ initially used because it wais similar to what Michel(1975) used and it was readily available in this laboratory , Sephadex G-100 During extract separation with Sephadex G-100, the flow rate of the column decreased significantly. The decrease is believed * to have been caused by particles in the venom extract which clogged up the nylon netting at the bottom of the column. The netting was acting as a filter to prevent the sephadex from flowing out of the column and into the fractions being collected. After a volume of ^8 ml was collected at 6°C, the column was run at room temperature. The fractions were monitored for protein content by spec tro photometric readings at 280 nm(Fig 15), The first part of this protein absorption curve is very similar in shape to that obtained by Michel(l975) on a Sephadex G-75 column, using a venom extract from Glycera convoluta. Because of the high degree of similarity, a second column wais run with Glycera dibranchiata venom. Due to the flow rate problem of Sephadex G-100, Sephacryl S-200 was chosen for the column bed. Several of the Sephadex G-100 fractions were tested for 51 Fig 15. Optical density of protein(O) and trypsin active(A) Sepliadex G-100 fractions of Glycera dibranchiata venom, run at 6 C, Protein collected at room temperature o (a). ^ o i e fVJ O o o 1>- o o o o o o o o o 0 rH >5 a 0 0 CO 4^ C! ir\ • 0 -H • ■HMO 4J C 00 ft (D rvj 0 T3 ^-^ 0 c trypsin activity (Table 6), The activity was plotted on the same "^ graph as the protein absorption(Fig 15). Maximum trypsin activity occurred between the two protein peaks. This corresponds to Michel's findings with Give era convoluta(1973). Michel did further studies on this protease enzyme to be sure that the enzyme was trypsin. However, it was found that the isolated enzyme did not act like trypsin on the ^-chain of insulin. There- fore, Michel refers to this enzyme as • trypsin- like' , possibly indicating a different type of trypsin. Due to this finding a closer examination of the trypsin isolated from Glycera dibran- chiata is needed. The Folin-Ciocalteu Assay was run on several Sephadex G-100 fractions, but the results were inconclusive because the stan- dards were not at a low enough concentration to correspond to the / samples. The Sephadex column was abandoned because of the decrease in flow rate which occurred during the extract separation. A second venom extract was run with Sephacryl S-200. This gel had a faster flow rate than the Sephadex gel, Sephacryl S-200 The Sephacryl column also had a slight decrease in flow rate when the extract was passed through. The flow rate went from 1 ,0 ml/rain to about 0.7 ml/min, however it was still much faster than the Sephadex gel. Four protein peaks were obtained from the extract (Fig 16). Their approximate molecular weight was calculated in the following manner, as described by Pharmacia, * By experimentally obtaining the elution volumes(Vg) of the stan- ( 33 Table 6. Trypsin determination of Sephadex G-lOO fractions. JLlg trypsin time(min) 0.20 ml of fraction # abs(255nm) timeCmin) ^.^ 0.427 1 0.051 ^.5 0.797 2 0.085 0.056 5 6 0.128 8 0.183 n 0.326 18 0.181 29 0.056 3^ Fig 16. Optical density of proteinC o ) and trypsin active (a ) Sephacryl S-200 fractions of Glycera dibranchiata venom, with 3,0 ml fractions collected at if min intervals at 6 C, 35 dard proteins, their K values were calculated; the formula for calculating K„( partition coefficient) is K-.,..= e" o , where V_, t o is the column void volume (which corresponas to the V of the Blue Dextran 2000), and V is the total bed voliime, ^ 2.1^,6 cm-^ for this column. Note that the V is the volume at which the highest concentration of a particular protein is collected. The K was plotted against the corresponding log molecular weight to obtain a linear relationship between elution volumes of the column and protein molecular weights. In this way the approxi- mate molecular weight of the extract protein peaks can be deter- mined (Fig 17). To compare these results with those of Michel (1975), the K values for Michel's Sephadex G-75 and G-200 were estimated using V =25,0 ml and V.-78,5 cm-^(Table 7), and plotted (Figs 18 & 19). This study revealed four protein peaks having molecular weights of >232 000, 160000, 121 000 and 31 000, corresponding to one high, two middle, and one low molecular weight protein peaks, Michel(1975) also obtained high, middle and low molecular weight protein peaks, as well as an additional intermediate peak in the Sephadex G-200 run, A more exact comparison cannot be made due to the lack of specific information available about Michel's columns. However, these results indicate a relationship could exist between the two venoms. To further compare these two venoms, the approximate mole- cular weight of the substance responsible for the trypsin activity was determined (Table 7. Figs 17-19). The first trypsin peak had a molecular weight of 200 000 , with the adjacent protein 36 Table 7. Molecular weight of venom protein peaks and trypsin activity for Give era dibranchiata from Sephacryl S-200 and for' Glycera convoluta from Sephadex G-75 and G-200 (Michel 1975). Protein Molecular Weight Glycera dibranchiata Glycera convoluta Venom peaks I 232 000 560 000- 232 000 ii 1 60 000 1 75 000- 55000 III 121 000 kk 000 IV 31 000 1 9 000- 10 500 Trypsin peaks A 200 000 88 000- 84 000 B 1 28 000 37 Fig 17. K V8 log protein molecular weight for Sephacryl S-200. Standard proteins(O), venom protein peaks (A), trypsin peaks(Lj). 38 1 Fig 18. K, vs log molecular weight for Michel' s(l 975) Sephadex G-75 coliimn. Standard proteins(O), venom protein peaks(A), trypsin peak([]). 0 faO •H :a u 0) H O s o ( 39 ') Fig 19, K vs log molecular weight for Michel's (1975) Sephadex G-200 column. Standard protein(o), venom protein peaks(A), trypsin peak(r]). 1 o o O o o O g O OD O O lA O ■P fA O X! O 00 ^ • > • 0 to 0 ui O ^0 peaks at >232 000 eind 160 000, Michel obtained this same se- quence with Sephadex G-75; a molecular weight of 84 000-88 000 for the trypsin peak, with protein peaks of about 2^0 000- 560 000 and 55 000-175 000, However, before any final conlusions are made, the trypsin fractions should be further tested using trypsin inhibitc:.^ and the ^ -chain of insulin, a^ Michel(1975) used. Selected fractions were assayed for toxicity. Since the extract had a solvent of buffer instead of sea water, a number of crabs were injected with an equivalent volume of pure buffer as a control measure. About 40% of these crabs stopped moving within 10-15 min. Therefore, no conclusions about the toxicity of the fractions could be made from this assay. Ammonium bicar- bonate was specifically used as the buffer for the chromatographic separation in this study so that the results could be directly compared to Michel' s( 1975). However, in order to determine the toxicity of the fractions, either a different buffer, which is not toxic to Uca, or a different test animal is needed, Michel used Daphnia by directly applying a small amount of extract to the exposed heart. The toxicity was measured as a function of the time it took for the Daphnia heart to stop beating, Daphnia was not used in this study because of the difficult technique required to expose the Daphnia. heart, and because they are fresh water animals ViThile the bloodworms are marine animals. It is biologically important to know what effect the venom has on the bloodworm's natural prey. Some of these fractions were also tested for protein con- centration according to the Folin-Ciocalteu assay. However, no conclusions could be made. 41 CONCLUSIONS The venom of Glycera dibranchiata appears to have at least two complementary components, one to increase the spreading of the venom inside the prey animal, and a lethal component, pos- sibly a neurotoxin* which paralyzes and/or kills the prey. From the Sephacryl S-Z:uO column four fractions were obtained with molecular weights of > 232 000, 160 000, 121 000 and 31 000. These four peaks correspond to four protein peaks obtained by Michel (1975), who used venom from Glycera convoluta on a Sephadex G-200 column, A trypsin-specific assay revealed two peaks of trypsin activity, having molecular weights of 200 000 and 120 000, The position of the first peak in relation to the protein peaks directly corresponds to the trypsin peak found by Michel(1975). This preliminary study on Glycera dibranchiata venom indicates that it is very close, if not the same, as Glycera convoluta venom. At least two more studies are needed to confirm the simi- larity. They are a more sensitive toxicity assay, such as the effect of the venom extract on the beating heart of a Daphnia (Michel 1975), and the effect of the trypsin substance in the venom on the ^ -chain of insulin. Results from these two studies would provide the needed information to fully confirm the simi- larity. Michel(1975) also did a collagenase assay. The assay is important because of the evidence of tissue hydrolysis upon injection of the venom in shrimp and marine fish(Curtis 1974, unpublished). The presence of collagenase would suggest that there is a component of the venom directly responsible for breaking down tissue and thereby increasing the rate at which ^2 the venom spreads. The presence of heat-resistant protein, revealed in this study, suggests a new area of research. It would be interesting to test the Sephacryl S-200 fractions for these specific proteins. It could reveal more about the specific components of Glycera dibraachiata venom. J J k3 SUMMAiff 1 . The purpose of this study was to isolate and chemically analyze the toxic principles of Glycera dibranchiata venom, 2. The crude venom extract was tested for toxicity, proteins, and trypsin content. The extract was fractionated by acetone precipitation and Sephadex G-lOO and Sephacryl S-200 column chromatography. 3. Acetone precipitation results suggested the presence of at least two toxic protein components, a spreading factor and a lethal factor, kt The crude extract was stable at -20°C for up to 35 days. At room temperature and above three protein components were found: a "quivering" factor, heat sensitive at rogm temperature and above, a lethal factor, heat sensitive at 100 C, for 10 min, and above, and a "lethargy" factor, heat resistant, 5, Four protein peaks and two trypsin-active peaks were obtained from Sephadex G-lOO and Sephacryl S-200 chromatography. The molecular weights of the four protein peaks were >232 000, 160 000, 121 000 and 31 000 , and the molecular weights of the trypsin-active peaks were 200 000 and 128 000, 6. These results indicate that G, dibranchiata venom is probably similar to G, convoluta venom, and more work on the toxic and trypsin-active principles, according to Michel 's(l 975) methods, is needed. kk LITERATURE CITED Clark, J.M. Jr., and Switzer, R.L. 196^. Experimental Biochemistry, W.H, Freeman and Company, San Francisco, California, Curtis, C. ^^7k. A study on the toxicity of the venom in Glycera dibranchiata. Unpublished, Ehlers, E, 1868, Die Borstenwiimer (Annelida Chaetopoda) nach Systematie^iier. und Anatomischen Untersuchungen, Wilhelm Engelmann, Leipzig, Freif elder, D, 1976, Physical Biochemistry and Molecular Biology, W.H, Freeman and Company, San Francisco, California, pp10-12, Ghiretti, F, I960, Toxicity of octopus saliva against Crustacea, Ann. N.Y. Aca, Sci,, 90:726-7^1. Halstead, B.W. 1959. Dangerous Marine Animals, Cornell Maritime Press , Cambridge , Halstead, B,W. 1965, Poisonous ajrid Venomous Marine Animals of the World, United States Government Printing Office, Washington, Hartman, 0, 1950, Goniadidae, Glyceridae and Nephtyidae, Kept, Allan Hancock Pacific Expiditions, 15: 1-1 81, Haurowitz, F, 1963, The Chemistry and Function of Proteins, Academic Press, New York, Heacox, A, 197^. The histology and histochemistry of the poison- gland complex in the polychaete Glycera dibranchiata. Thesis, Adelphi University, 66pp, University Microfilms, Ann Arbor, Mich, Klawe, W.L,, and Dickie, L.M. 1957. Biology of the bloodworm, Gl.yce;?a dibranchiata Ehlers, and its relation to the blood- worm fishery of the Maritime Provinces, Fish, Res, Board Can, Bull., 115:1-37. Michel, C. 1966. Machoires et glandes annexes de Glycera convo- luta, annelide polychete Glyceridae. Cah, Biol,, March BT3^7-373. Michel, C,, and Keil, B, 1975. Biologically active proteins in the polychaetous annelia, Glycera convoluta Keferstein,. Comp, Biochem, Physiol,, 50B: 29-33. Michel, C, and Robin, Y, 1972, Premieres donnees biochimiques sur les glandes k venin de Glycera convoluta Keferstein, C, R. Seanc. Soc. Biol., 166:853-857. Neilands, J.B. 196if. Outline of Enzyme Chemistry. John Wiley and Sons, Inc., New York, pi 27. ^5 Ockeiraann, K.W. and Vahl, 0. 1970. On the biology of the polychaete Glycera alba, especially its burrowing and feeding, Ophelia, 8:275-2%. Rick, W, 1965. Trypsin, ed, by Pfens-Ulrich Bergmeyer in Methods of Enzymatic Analysis, p8l5-8l8, Roberts, D.V, 1977, Enzyme Kinetics. Cambridge University Press, New York. pplOAf-106. Schwert, G.W. and TaJienaka, Y. 1955. ^ spec tro photometric determination of trypsin and chymotrypsin, Biochim, et Biophys. Acta., 16:570-575. ( 96912YF ™ i!/2S/94 45190 »4tsil DATE DUE GAYLOBO PRINTED IN us A SnET ?**?(•* COlLECr LIERARY SWEET P^I^S.Va 245S5