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Byline: H. Waheed, S. K. Doman and A. Ahmed


The hemoglobin from Leaf-nosed Viper (Eristocophis macmahonii) was analyzed to better understand interspecies relationship among various snakes at the molecular level. After isolation, the hemoglobin from erythrocytes was analyzed by cation-exchange chromatography. The chains from hemoglobin were separated by reversed phase-high performance liquid chromatography. The N-terminal amino acid sequences of globin chains were obtained by the process of Edman degradation in an automated protein sequencer using an online Phenylthiohydantoin (PTH) analyzer. The obtained sequences were compared using online pairwise sequence alignment tool and Clustal Omega. The N-terminal protein sequence results revealed that two hemoglobin components, comprised of [alpha]A, [beta]I and [beta]II-globin chains are expressed in Leaf-nosed viper.

The [alpha]A-globin chain showed highest similarity with Sindhi krait, [beta]I with Texas indigo snake and [beta]II-with Indian cobra and interestingly with Blue-lipped sea krait (sea snake). N-terminal sequence alignment studies also showed some important amino acid substitutions that may affect iso-Hb composition and function.

Keywords: Eristocophis macmahonii; hemoglobin characterization; evolution; amino acid sequence; snakes; Leaf-nosed viper.


Throughout the evolution, hemoglobin has played an important physiological role of transporting oxygen and carbon dioxide. They were evolved from stem lineage of jawed vertebrates (gnathostomes) because of two genome duplication events (Stroz, 2016). The globin proteins retain their specific function in oxygen transport and storage to aid physiological division of labor during developmental stages of larger vertebrates. Hemoglobin had been selected evolutionary to function as oxygen transport protein due to its allosteric and cooperative oxygen delivery and uptake (Stroz et al., 2013; Burmester and Hankeln, 2014). It serves as a bi-directional respiratory carrier by; binding oxygen in lungs or other respiratory organs and unloading at metabolizing sites and returning carbon dioxide to the respiratory organs (Marengo-Rowe, 2006).

This universal oxygen transport protein is a tetramer composed of a pair of identical [alpha]-and [beta]-globin chains that arise from duplications of an ancestral hemoglobin gene into two different paralogous gene families. These paralogous genes further duplicate to give [alpha]-and [beta]-globin to form isomeric Hbs (iso-Hbs). The expression of different iso-Hb is the adaptation of vertebrates to fulfill energy demands during various developmental stages. Their differential expression is regulated by various physiological and ontogenetic factors (Lukin and Ho, 2004; Hardison, 2012). The difference in their primary structure has resulted in altering the ligand affinity and sensitivity towards allosteric molecule, thus altering the function. This expression versatility enables the fulfillment of oxygen at the various developmental stages of an organism (Brittain, 2002).

Reptiles (snakes, lizards, and amphisbaenians) constitute phylogenetically diverse group with more than 9400 extant species (Wiens et al., 2012; Pyron et al., 2013). Due to their characteristic physiological system, the reptiles in the snake family with 3070 extant species, serve as an important model to better understand its behavior and molecular evolution (Vidal and Hedges, 2009). Snakes being ectothermic, experience variation in oxygen affinity. Also, their oxygen affinity is affected by the changes in other environmental conditions and physiological states (Herman and Ingermann, 1996; Amiel et al., 2011; Bovo et al., 2015). The present goal of the study was to characterize the hemoglobin of Leaf-nosed viper (Eristicophis macmahonii), member of family Viperidae. The snake is found in the deserts of Afghanistan, Iran and Pakistan. In Pakistan, it is mainly found in Nushki, Kharan and other desert areas of Balochistan (Khan, 2002).

These snakes experience high summer temperature (40-50 AdegC) along with diurnal temperature variation. Various physiological and behavioral thermoregulatory mechanisms have been adopted by them to modulate their body temperature to meet their metabolic needs (Girons, 1980; Seebacher and Franklin, 2005). The only globin chain sequence reported from Viper snakes is from Vipera aspis describing the sequence of [alpha]-chain of hemoglobin (Duguet et al., 1974). Here we describe the preliminary studies on hemoglobin of Leaf-nosed viper.


Blood Sample Collection and Hemolysate Preparation: Leaf-nosed viper (Eristicophis macmahonii) specimens were collected from Nushki, Balochistan, Pakistan. The blood was collected from the anesthetized snake by cardiac puncture in the presence of heparin as an anticoagulant. The blood samples were centrifuged to separate erythrocytes from the plasma. The erythrocytes were then washed with cold 0.9% NaCl solution (saline). The washed cells were subjected to hypotonic lysis. The hemolysate (supernatant) after dialysis against deionized water was lyophilized and stored (Riggs, 1981).

Isolation of Hemoglobin Components: Cation exchange column (Mono S-5/50 GL) was equilibrated with 20mM sodium phosphate buffer, pH 6.0 containing 0.01% KCN. The crude hemolysate was subjected to the column and components were eluted using a gradient of 0-0.5 M NaCl in 15 min. at a flow rate of 1 mL/min. The absorbance of the eluent was recorded at 280 and 415 nm.

Globin Chain Separation: The globin was precipitated in cold acidified acetone. The pellets were then washed with cold acetone, solubilized and dialyzed against deionized water and lyophilized (Rossi-Fanelli and Antonini, 1958). The globin chain separation was carried out by using a reversed phase column (Nucleosil C4-250 x 4.6 mm) equilibrated in solvent A (0.1% TFA in water). Globin (1mg/mL), dissolved in solvent A was loaded to the column. Globin chains were eluted with solvent B (0.1% TFA in acetonitrile) using a linear gradient of 37-45% in 60 minutes, with a flow rate of 1mL/min. The absorbance recorded at 280 nm.

Amino Acid Sequence: The N-terminal amino acid sequence of purified globin chains was deduced by Edman degradation in an automated sequencer; model Procise 491 (Applied Biosystems). The partial amino acid sequence of LNV globin chains established up to 30 residues, were compared with other reported snakes globin sequences using online Pairwise sequence alignment tool ( and the multiple sequence alignment by Clustal Omega (


Purification of Hemoglobin: Adult Leaf-nosed viper (E. macmahonii) hemolysate that was analyzed by cation exchange chromatography resolved into two peaks. The major (P1) and minor (P2) peaks were observed in the ratio of 1:0.3 (Fig. 1). The crude globin was resolved into 3 major peaks by RP-HPLC (Fig. 2). The RP-HPLC peaks eluted at 32, 51, 55 and 56 min were annotated as [beta]II, [beta]I, [alpha]A and [alpha]A globin chains respectively based on N-terminal amino sequence (Fig. 2). Based on these observations, it is expected that at least two iso-Hb expressed in adult leaf-nosed viper and the major iso-Hb is adult type i.e. HbA.

N-terminal Amino Acid Sequence

[alpha]A globin chain: The N-terminal amino acid sequence of intact [alpha]A globin chains of Leaf-nosed viper (E. macmahonii) was successfully established up to thirty amino acid residues by Edman sequencing. Based on N-terminal sequence analysis, Leaf-nosed viper [alpha]A chain showed maximum sequence similarity (93.3%) with [alpha]A chain from Sindhi krait (Bungarus sindanus sindanus), followed by Indian cobra (Naja naja), Texas indigo snake (Drymarchon melanurus erebennus) and Black-banded sea krait (Laticauda semifasciata) where 90% similarity was observed. Blue-lipped sea krait (Laticauda laticaudata) showed 86.7%, Small-headed sea snake (Microcephalophis gracilis) and Eastern diamondback rattlesnake (Crotalus adamanteus) showed 83.3% similarity. Aspic viper (Vipera aspis) that belongs to viperidae family showed least similarity i.e. 78.8% as shown in Fig: 3.

[beta]I globin chain: The [beta]I globin chain sequence of Leaf-nosed viper showed highest similarity (93.3%) with [beta]I chain of Texas indigo snake (Drymarchon melanurus erebennus). It also showed 90% similarity with Eastern diamondback rattlesnake (Crotalus adamanteus), 80% with Blue-lipped sea krait (Laticauda laticaudata) and Water snake (Liophis miliaris). Least similarity was observed with Black-banded sea krait (Laticauda semifasciata) and Indian cobra (Naja naja) i.e. 73.3% and 70% respectively (Fig. 4).

[beta]II globin chain: The [beta]II globin chain of Leaf-nosed viper showed maximum sequence similarity (93.3%) with [beta] chain sequence from Indian cobra (Naja naja) and Blue-lipped sea krait (Laticauda laticaudata). Similarity of 86.7% was observed with both Small-headed sea snake (Microcephalophis gracilis) and Eastern diamondback rattlesnake (Crotalus adamanteus). Least similarity of 83.3% was observed with Black-banded sea krait (Laticauda semifasciata) [beta] globin chain (Fig. 5).

Multiple Sequence Alignment

[alpha]A globin chain: Multiple sequence alignment of the of [alpha]A globin chain of Leaf-nosed viper is shown in Fig. 3. The sequence comparison showed some important amino acid substitutions in Leaf-nosed viper [alpha]A globin chain that includes the presence of Ser at position 3, in the LNV while majority of other species have Thr. At position 8, Asn is present in LNV while in most species compared, showed the presence of Ala or Ser or Thr. At position 13, Ala is present in Leaf-nosed viper, and few other snakes, while Ser is present in some snake species Further, it was observed that Ser at position 15 in LNV is replaced by Ala, Val or Gly in other species. The residue at position 18 in LNV identified is Gly, while in others snake Ser is present. At position 21, Pro is observed in Leaf-nosed viper and Aspic viper, while in all other species Ala is present. The highest sequence similarity in first 30 amino acid residues is 93.3% with Sindhi Krait and least similarity 78.8% with Aspic viper.

[beta]I globin chain: Multiple sequence alignment of [beta]I globin chain of Leaf-nosed viper is shown in Fig. 4. At position 9, Ser is present in Leaf-nosed viper and some other species while in others Asn is present. In Leaf-nosed viper and Eastern diamondback rattlesnake Thr is at position 10, which is replaced with Ala or Ile in other snakes. Thr or Ser at 12th position mostly is replaced with Asn in Eastern diamondback rattlesnake and Indian cobra. Ala at position 13 in Leaf-nosed viper, Eastern diamondback rattlesnake and Water snake is replaced mostly with Ser in others. At position 21, Pro is present in Leaf-nosed viper, Eastern diamondback rattlesnake and Texas indigo snake is replaced with Ala or Gly in others. Ala at position 22 is replaced with Gln in Blue-lipped sea krait, His in Black-banded sea krait and Glu in Indian cobra.

Ser is present at position 29 is replaced with different amino acids i.e. Ala, Gly, Cys or Lys. Asn is present at 30 position in LNV, while it is also replaced by Arg except Gln in Eastern diamondback rattlesnake and Leu in Indian cobra. In contrast to [alpha] chain, the [beta] chain of Leaf-nosed viper has highest similarity 93.3% with Texas indigo snake and least similarity 70% with Indian cobra.

[beta]II globin chain: Multiple sequence alignment of [beta]II globin chain is tabulated in Fig. 5. At position 14, Phe is present in Leaf-nosed viper while in all other compared snakes, Leu is present. At 16 position, Ser in Leaf-nosed viper and Blue-lipped sea krait is replaced with Ala, Gly or Pro in rest of the compared species. At position 21, Pro in Leaf-nosed viper, Indian cobra and Eastern diamondback rattlesnake is replaced with Ala in other snakes. In contrast to [beta]I chain, the [beta]II chain of leaf-nosed viper has highest similarity 93.3% with Indian cobra and least similarity 83.3% with Black-banded sea krait.


Two or more structurally different iso-Hbs are expressed during various developmental stages of squamate reptiles as a regulatory mechanism. The expression and concentration of different iso-Hbs present depend upon their metabolic need of oxygen that differs during different stages of development as well as various physiological states (Storz and Moriyama, 2008; Storz et al., 2011). These iso-Hbs are classified into Hb-D or Hb-A based on type of [alpha]-globin ([alpha]D or [alpha]A) present in that specific iso-Hb. Phylogenetic studies of hemoglobin showed that, single copy of [alpha]D or [alpha]A-globin and ss-globin gene duplicates i.e. [beta]I and [beta]II are expressed in adult snakes (Bonilla et al., 1994; Gorr et al., 1998; Hoffmann et al., 2010; Storz et al., 2015). Mainly, two hemoglobin components and four globin chains have been observed in snakes (Gorr et al., 1998; Storz et al., 2015).

These include: Blue-lipped sea krait (Eguchi and Eguchi, 2002), Indian cobra (Naqvi et al., 1994), Black-banded sea krait (Eguchi and Eguchi, 2003), Small-headed sea snake (Islam et al., 1990), Indigo snake (Stoeckelhuber et al., 2005) and Indian Python (Gorr et al., 1998). Expression of three iso-Hbs were observed in SouthAmerican rattlesnake and Sindhi krait where at least one of the iso-Hbs is expected to be the post-translational modified form of the major iso-Hb present (Storz et al., 2015; Waheed et al., 2016). Our results showed that at least two types of hemoglobin components are expressed in Leaf-nosed viper. One of the two components are expected to be adult type hemoglobin i.e. Hb-A. The other component could be the isoform of the adult type and may be a physiological adaptation of this snake to its environment. In RP-HPLC chromatogram, a small peak was also observed at a retention time of 40-45 min., which showed the mix type of sequence (data not shown).

It is already being reported that several snake hemoglobin, at high pH, undergo oxygenation-linked dissociation into [alpha][beta]-dimers in the absence of ATP (organic phosphate) (Storz et al., 2015). The mixed type of sequence that was observed could be possibly due to the dimeric form of hemoglobin. However, these were our initial results and further experimental authentication is in progress.

Conclusion: From our initial experimental observations, we conclude that two hemoglobin components that are composed of [alpha]A, [beta]I and [beta]II-globin chains, are expressed in Leaf-nosed viper. The globin chains were designated as [alpha]A, [beta]I and [beta]II based on their N-terminal sequence alignment analysis. Multiple sequence alignment studies showed some interesting substitutions that may influence its iso-Hb composition and function. Further studies on hemoglobin from this snake are in progress and will be reported elsewhere. Currently, only the complete amino acid sequence of an [alpha]-chain of Viper (Vipera aspis) a member of family Viperidae hemoglobin is available (Girons, 1980). The knowledge gained from this study will provide an insight into the understanding of the evolutionary relationships and molecular mechanisms of physiological adaptation among various snakes.

Acknowledgements: The HW is grateful to Prof. Dr. Atta-ur-Rahman (FRS) and Prof. Dr. M. Iqbal Choudhary, Director, H.E.J. Research Institute of Chemistry, University of Karachi for their guidance and financial support. The travel grant to HW was provided by Dr. Zafar Zaidi Memorial Trust. We also acknowledge Dr. Rukhsana Lalani of Chapman University School of Pharmacy for proof reading of this manuscript. This research was made possible using Analytical Core Facility, Chapman University School of Pharmacy.

Conflict of Interest: The authors declare that they do not have any conflicts of interest with the contents of this article.


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Publication:Journal of Animal and Plant Sciences
Date:Dec 31, 2018

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