Enzyme activities and biological functions of snake venomsMarshall D. McCueDepartment of Ecology and Evolutionary Biology, University of California, Irvine, CA 92617-2525,USACurrent address: Department of Biology, University of Arkansas, Fayetteville, AR 72701, USA.e-mail: email@example.comAbstract. While snake venoms are well characterized pharmacologically, the evolutionary history andfunctional utility of snake venoms has not been thoroughly investigated. Hypotheses advanced overthe past hundred years suggest that snake venoms may increase evolutionary fitness by facilitatingone or more of three functions; prey capture, defense, and digestion. This review provides a newapproach to analyzing these hypotheses by reviewing patterns of venom enzyme activity fromover one hundred species of venomous snakes. The patterns uncovered suggest that venoms foundamong the subfamilies, Elapinae, Viperinae, and Crotalinae do not statistically differ with regardto phospholipase A 2 , phosphomonoesterase, and phosphodiesterase activities. While this findingsupports theories that venomous snakes may have evolved from a single common ancestor, viperineand crotaline venoms are shown to have proteolytic activities (e.g. L-amino acid oxidase, nonspecificendopeptidase, and trypsin-like activity) that are dramatically higher than elapine venoms. Theproteolytic activities of venoms are significantly higher in the more recently derived crotalines,suggesting that the evolution of these venoms may be influenced by their digestive function. Thisreview also introduces a novel index for comparing ‘toxic risk’, a measure that takes into accountvenom yield as well as toxicity. The results suggest that the toxic risk of these three subfamilies donot differ significantly despite their distinct evolutionary histories. This review also identifies taxawhose venom enzyme activities are dissimilar from their confamilials. Because of their unique venomproperties, taxa whose venom greatly differs from their confamilials may prove useful for testingforthcoming hypotheses about the biological function and evolutionary history of snake venoms.Key words: Elapidae; enzymes; evolution; function; phylogeny; snake; toxic risk; toxinology; venom;Viperidae.IntroductionSnake venoms are among the best pharmacologically characterized natural toxins,chiefly because of their deleterious effects on humans. While these complex, proteinrich mixtures have been extensively separated and fractionated for over half a century,our understanding of the evolution of venomous snakes has relied on compar-© Koninklijke Brill NV, Leiden, 2005 APPLIED HERPETOLOGY 2: 109-123Also available online - www.brill.nl
110 Marshall D. McCueative morphology (Vidal, 2002; Jackson, 2003) and molecular genetics (Kraus andBrown, 1998; Slowinski and Lawson, 2002). Unfortunately, genetic and morphologicalanalyses alone cannot provide much evidence regarding evolution of venomcomponents, and offer no insight into the evolutionary and biological utility of snakevenoms. This study has two primary functions: (1) to review and summarize theexisting body of toxinological literature regarding the enzyme activities of snakevenoms, and (2) to encourage applied researchers to consider the natural functionsand selective forces that have shaped snake venoms over evolutionary time. Thesefindings should be of particular interest to applied toxinological researchers whodeal with these intriguing mixtures exclusively at the pharmacological level.While the primary biological utility of snake venoms is not well understood froman evolutionary perspective, this has not prevented naturalists from speculatingabout venoms’ biological utilities for over one hundred years (Mitchell, 1868;Shortt, 1870). Most of these hypotheses regarding the function of snake venomshave focused on three adaptive advantages: prey capture, defense, and digestion.Whether a result of difficulties associated with experimental design or the obviousconnection between a snake bite and death, very few scientific researchers haveattempted to investigate the evolutionary utility of snake venoms (Thomas andPough, 1979; Daltry et al., 1997; Andrade and Abe, 1999; McCue, 2002).Evolution of venomous snakesVenomous snakes are a polyphyletic group of Colubroidea that includes all familymembers of Elapidae and Viperidae, and some of the members of the familiesAtractaspidae and Colubridae. Because of the difficulties in definitively identifyingwhich snakes belonging to the families Atractaspidae and Colubridae are venomous(Vidal, 2002), and because venomous atractaspid and colubrid snakes are so poorlyrepresented in the toxinological literature (Rodriguez-Robles, 1994; Weinsteinand Kardong, 1994), these groups are not further addressed here. This reviewfocuses on the venom characteristics of the three most widely researched venomoussubfamilies: the Elapinae (Family Elapidae) and the Viperinae and Crotalinae(Family Viperidae). These lineages are believed to have originated in the Miocene,but remain sparsely represented in the fossil record (Nilson and Andren, 1997; Rage,1997). Like their fossil record, scientific discussions concerning the evolutionaryand selective forces responsible for shaping their venoms are scant.Several studies have shown that the composition of snake venom is geneticallycontrolled, and thus subject to evolution via natural selection like any heritable trait(Jimenez-Porras, 1964; Aird et al., 1989). Therefore, it should be possible to makeevolutionary inferences based on the current patterns of venom phenotypes. Thispaper examines patterns in venom protein content, toxicity, and yield, and comparesspecific enzyme activities among over one hundred venomous snake species fromthree subfamilies. The purpose of this investigation is to uncover patterns in thechemical activities and composition of venoms. Such patterns can then be used to
Enzyme activities and biological functions of snake venoms 111address the long-standing hypotheses about the biological function and evolutionaryradiation of snake venoms.Comparing chemical activities of snake venomsSnake venoms are complex mixtures composed chiefly of varied enzymatic andnonenzymatic toxins. Although a single snake venom sample may contain dozensof enzymatic toxins, these enzymes are generally grouped into a few classes by toxinologists.The most commonly quantified classes of snake venom enzymes includephospholipase A 2 (PLA 2 ), phosphodiesterase, phosphomonoesterase, L-amino acidoxidase, specific endopeptidases, and nonspecific endopeptidases (Iwanaga andSuzuki, 1979). The specific activities of each of these can be measured using differentsubstrates.Most comparative studies of snake venoms do not quantify enzyme concentrationdirectly, but rather, measure venoms’ specific activities on various molecularsubstrates. Because the enzyme composition of particular venom fractions can varywidely (Boumrah et al., 1993; Komori et al., 1995), measurements of specificactivity of whole venoms are employed in these analyses. Furthermore, manyvenom components are believed to work synergistically with each other, or withcomponents of prey tissue (Teng et al., 1984; Tan and Armugam, 1990), and thusfractionated venoms offer a less complete collection of potential synergies. As aresult, this study references only toxinological studies that use either fresh venomsor freshly reconstituted whole venoms. Reconstituted venoms are most commonlyused in laboratory research and are well known to be pharmacologically equivalentto fresh venoms (Minton and Weinstein, 1986; Hayes et al., 1995).Several studies have demonstrated that venom from conspecific snakes can varyontogenetically (Bonilla et al., 1973; Meier and Freyvogel, 1980; Meier, 1986;Andrade and Abe, 1999), seasonally (Gregory-Dwyer et al., 1986), interdemically(Aird, 1985; Minton and Weinstein, 1986; Wilkinson et al., 1991; Rodrigues et al.,1998), and with physical condition (Klauber, 1997). Because of the numeroussources of qualitative and quantitative variation among venoms, this study drawsfrom a broad range of primary sources to explore patterns in toxinological pharmacologicalproperties of a diverse collection of snake venoms.MethodsBiochemical and toxinological literature were reviewed for quantitative datasetscomparing specific activities of various venom enzymes. For this study a datasetof 109 species representing three subfamilies was compiled (Zeller, 1948; Deutschand Diniz, 1955; Richards et al., 1965; Oshima and Iwanaga, 1969; Passey, 1969;Mebs, 1970; Kocholaty et al., 1971; Pereira et al., 1971; Geiger and Kortmann,1977). To minimize statistical bias, only toxinological studies comparing venomenzymes in at least five snake species are referenced and analyzed; most of these
112 Marshall D. McCuestudies examined more than 20 species from the three subfamilies. Although manyof these studies examined the enzyme activities on similar substrates, each studyvaried slightly in experimental conditions including venom concentration, substratepreparation, and experimental temperature. To correct for absolute differences inenzyme activities that may be associated with these experimental differences, thespecific enzyme activities from each study were first normalized.Normalization involved assigning a maximum value of 100 to the venom samplewith greatest enzyme activity from each series of experiments. Specific enzymeactivities for each snake species in these studies were converted to proportionsof this value (ranging from 0 to 100). These normalized values were then log 10transformed to minimize the effects of limited sample size on statistical evaluations.These log 10 transformed venom enzyme activities were compared within andamong snake subfamilies using model II analysis of variance (ANOVA) andBonferroni corrections for multiple comparisons. Post hoc comparisons betweenthe mean values among subfamilies were conducted using Fisher’s PLSD with asignificance of p = 0.0167. Comparisons of enzyme activity within subfamilieswere conducted using one-tailed t-tests. All analyses were calculated using StatViewsoftware (SAS).An index termed ‘toxic risk’ was also developed. This index considered both thetoxicity and venom yield from each snake species. Venom yield was estimated byaveraging values of dry venom yield reported in literature for various snake species(Kocholaty et al., 1971). The toxicity (LD 50 ) was also averaged from values reportedin literature. LD 50 values used in these analyses were reviewed in Brown (1973) andMebs (1978). Only intraperitoneal (IP) and intramuscular (IM) LD 50 values wereused in these analyses; if both values were available, they were averaged. Althoughsnake venoms are generally most toxic when administered intravenously (IV), suchmeasures of toxicity were not used as snake venom injected directly into a majorvein may be biologically unrealistic. Reciprocals of the LD 50 values were multipliedby mean dry venom yields for each species resulting in a ‘toxic risk’ index. Thesevalues were then normalized and log 10 transformed as described above.ResultsProtein content of the venoms of the three subfamilies (Elapinae, Viperinae, andCrotalinae) varied significantly (p = 0.0008; fig. 1a). While there was no differencebetween the protein content of viperine and crotaline venoms, elapine venomscontained significantly more protein than both viperine (p = 0.0036) and crotalinevenoms (p = 0.0003; table 2). The toxic risk index showed no significant differencebetween the three subfamilies (p = 0.5116; table 1; fig. 1b).Levels of PLA 2 (p = 0.2117), phosphodiesterase (p = 0.5964), and phosphomonoesterase(p = 0.2120) activities were statistically indistinguishable amongthe three subfamilies (table 1; fig. 2; fig. 3a). Nevertheless, significant differences inspecific enzyme activities among these subfamilies were revealed.
Enzyme activities and biological functions of snake venoms 113Figure 1. (a) Percentage of protein in dried Elapinae, Viperinae, and Crotalinae venoms. Data adaptedfrom Kocholaty et al., 1971. (b) Relative toxicity (toxic risk) of snake venoms calculated as the log 10transformed product of mean dried venom yield and the reciprocal of mean LD 50 (IM and IP) reportedin literature (Brown, 1973; Mebs, 1978).
114 Marshall D. McCueTable 1. Results of ANOVAs comparing log 10 transformed normalized enzyme activities and othervenom characteristics between snake subfamilies. Significance is set at p < 0.0167 for multiplecomparisons. Significant values are presented in boldface.Enzyme df Sum of Mean F p Lambda Poweractivity squares squarePhosphomonoesterase 2 0.188 0.094 0.520 0.5964 1.040 0.130Residual 88 15.879 0.180Phosphodiesterase 2 0.688 0.344 1.582 0.2120 3.163 0.314Residual 80 17.406 0.218Phospholipase A 2 0.778 0.389 1.596 0.2117 3.191 0.313Residual 57 13.892 0.244L-amino acid oxidase 2 7.625 3.812 17.951
Enzyme activities and biological functions of snake venoms 115Figure 2. (a) Normalized and log 10 transformed phospholipase A 2 activity of Elapinae, Viperinae, andCrotalinae venoms. Data is adapted from multiple toxinological studies (Mebs, 1970; Kocholaty et al.,1971). (b) Normalized and log 10 transformed phosphodiesterase activity of several snake venoms.Data is adapted from several toxinological studies (Richards et al., 1965; Mebs, 1970; Kocholatyet al., 1971; Pereira et al., 1971).
116 Marshall D. McCueFigure 3. (a) Normalized and log 10 transformed phosphomonoesterase activity of Elapinae, Viperinae,and Crotalinae venoms. Data is adapted from several toxinological studies (Richards et al., 1965;Mebs, 1970; Pereira et al., 1971). (b) Normalized and log 10 transformed L-amino acid oxidase activityof several snake venoms. Data is adapted from several sources (Zeller, 1948; Mebs, 1970; Kocholatyet al., 1971).
Enzyme activities and biological functions of snake venoms 117Nonspecific endopeptidase activity also differed among elapine, viperine, andcrotaline venoms (p < 0.0001; table 1). Viperine venoms (p < 0.0001) andcrotaline venoms (p < 0.0001) demonstrated greater nonspecific endopeptidaseactivity compared to elapid venoms (table 2). Nonspecific endopeptidase activity ofcrotaline venoms was over 2-fold greater than viperine venoms and 10-fold greaterthan elapine venoms (fig. 4a).Specific endopeptidase activity (trypsin-like and chymotrypsin-like) also differedamong the three subfamilies (p < 0.0001; table 1). Trypsin- and chymotrypsinlikeactivities were significantly greater in the crotaline venoms than the othersubfamilies (p
118 Marshall D. McCue
Enzyme activities and biological functions of snake venoms 119Venom utilityAn early hypothesis about the biological significance of snake venom suggested thatit functioned exclusively for prey capture (Githens, 1935). Although this hypothesisseems obvious, it has not undergone rigorous experimental testing (e.g. no studyhas examined whether venomous snakes can capture prey equally well without thefunction of their venom delivery system). Despite the lack of quantitative support forthe prey capture hypothesis, similar claims can be found in recent literature (Saint-Girons, 1997; Kini and Chan, 1999; Sasa, 1999). Not only do the vast majorityof nonvenomous snakes capture prey through constriction, but the ancestral stock ofvenomous snakes also undoubtedly captured prey by constriction (Rage, 1997). Onestudy even found that the frequency of prey constriction among venomous elapineswas statistically identical to that observed in nonvenomous snakes (Shine, 1985)If the primary role of venom was for prey capture, one might expect to see a correlationbetween relative toxicity and prey type (Sasa, 1999). Several studies haveinvestigated whether such relationships exist. While some of these report a correlationbetween toxicity and prey type (Daltry et al., 1996, 1997), others have failed tofind any correlations (Soto et al., 1988; Andrade and Abe, 1999). Furthermore, somespecies that have evolved to specialize on inanimate or slow-moving prey, such asegg and insect eating elapines, possess (apparently unnecessarily) potent venoms.Finally, if venom had evolved exclusively for prey capture, one might predictthat venoms of elapine snakes, which generally prey upon ectotherms (Mushinsky,1987), would have statistically distinct toxic risk (fig. 1b), particularly since thesedata are based on toxicity in evolutionary naïve laboratory mice. Prior to thisanalyses, one might have expected the venoms of snakes evolved for immobilizingendothermic prey (i.e. viperines and crotalines) to have higher toxic risk; this wasnot the case.Some reports suggest that snake venoms evolved as a defensive weapon (Williamset al., 1988; Davidson and Dennis, 1990; Underwood, 1997). This hypothesis hasfound recent support by observations that the amount of venom expended duringdefensive strikes in rattlesnakes was 10-fold that of prey strikes (Young and Zahn,2001). It was also determined that secondary strikes by a crotaline rattlesnakedelivered only 40% of the venom delivered by a primary strike (Hayes, 1992).While this study is unable to determine whether this phenomenon directly resultsfrom reduced venom inventory during a second strike, it is likely that venom usedin defensive activity could limit its utility for subsequent prey capture, particularlywhen one considers the lengthy time (several days) required to replenish the venomFigure 4. (a) Normalized and log 10 transformed nonspecific endopeptidase activity of Elapinae,Viperinae, and Crotalinae venoms. Data is adapted from several sources (Deutsch and Diniz, 1955;Richards et al., 1965; Oshima and Iwanaga, 1969; Mebs, 1970; Geiger and Kortmann, 1977).(b) Normalized and log 10 transformed trypsin- and chymotrypsin-like activity of several snakevenoms. Data is adapted from several sources (Deutsch and Diniz, 1955; Oshima and Iwanaga, 1969;Passey, 1969; Mebs, 1970; Kocholaty et al., 1971; Geiger and Kortmann, 1977).
120 Marshall D. McCuecache (Kochva et al., 1975). One study suggested that the amount of venomdischarge be ‘metered’ depending on prey size (Hayes et al., 1995), however itremains unclear whether this finding supports either the prey capture or the defensehypothesis.Venom has long been hypothesized to facilitate digestion in snakes (Zeller,1948). Like the two previous hypotheses the digestion hypothesis is also frequentlymentioned in current literature (Daltry et al., 1996; Anderson and Ownby, 1997;Kini and Chan, 1999; Sasa, 1999; Vidal, 2002), but often with scant empiricalevidence. It probably stems from observations that virtually all enzymes found insnake venoms are functionally and structurally similar to the digestive secretionsfound among other vertebrates (Yun et al., 1998). So far, two studies have foundevidence that crotaline venoms influenced rates of digestion and gut passage(Thomas and Pough, 1979; McCue, 2002). Evidence for the digestive role ofvenoms is also provided by the extensive tissue damage that typically follows asnakebite, much of which does not occur until several minutes after envenomation.Some studies even demonstrate tissue digestion continuing several hours followingenvenomation (Mebs and Ownby, 1990; Rodriguez-Acosta et al., 1999).If venoms were evolutionarily selected to complement the digestive process insnakes, one might expect to see the greatest proteolytic activities within snakespecies that tended to consume largest relative prey masses. While elapines generallyingest meals that are less than 20% of their body mass, much larger meals(i.e. greater than their own body mass) are occasionally consumed by viperinesand crotalines (Greene, 1983). This fact, in light of the dramatically higher proteolyticenzyme activities in viperine and crotaline venoms, supports the likelihoodthat snake venom serves some digestive role.Despite the apparent intuitiveness and long history of discussion on snake venoms,we should be cautious with claims regarding their primary biological utility.While this study of enzyme activity finds support for the digestive hypothesis, it isvery likely that snake venoms are multifunctional, and may differ based on speciesspecificevolutionary pressures. As we increase our understanding of the utility thatvenoms provide snakes, we can more effectively develop medical applications forthese powerful mixtures.Acknowledgements. I would like to thank A.F. Bennett and T.J. Bradley forhelpful comments on the manuscript. Thanks also to W. Wüster for his constructivecomments during his review. I would finally like to acknowledge NSF GraduateResearch Fellowship awarded to M.D. McCue and NSF IBN-0091308 awarded toA.F. Bennett and J.W. Hicks.
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