- Email: [email protected]
Differential sensitivity of Chironomus and human hemoglobin to gamma radiation
Biochemical and Biophysical Research Communications 476 (2016) 371e378
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Differential sensitivity of Chironomus and human hemoglobin to gamma radiation Pallavi S. Gaikwad a, b, Lata Panicker c, Madhura Mohole d, Sangeeta Sawant d, Rita Mukhopadhyaya b, Bimalendu B. Nath a, * a
Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007, India Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India d Bioinformatics Center, Savitribai Phule Pune University, Pune, 411007, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2016 Accepted 25 May 2016 Available online 26 May 2016
Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. © 2016 Elsevier Inc. All rights reserved.
Keywords: Chironomus hemoglobin Gamma radiation tolerance Human hemoglobin Heme-binding pocket analysis
1. Introduction Hemoglobin (Hb) constitutes a remarkable family of genes and proteins present across all phyla. It has a long history of 600e800 million years of evolution [1]. Hemoglobins (Hbs) are intracellular in jawed vertebrates, found exclusively in erythroid cells in sharp contrast to rarely encountered invertebrate animals where Hbs are extracellular [2,3]. In adult human, Hb is a heterotetramer, consisting of two a-globins and two b-globin polypeptides conjugated with their heme groups [4]. Down the evolutionary line, one can encounter an ancient group of dipteran insects, the chironomid midges that evolved approximately 200 million years ago and are the only insects possessing Hb in the hemolymph conferring
* Corresponding author. Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule Pune University, Pune, 411 007, India. E-mail addresses: [email protected], [email protected] (B.B. Nath). http://dx.doi.org/10.1016/j.bbrc.2016.05.129 0006-291X/© 2016 Elsevier Inc. All rights reserved.
characteristic red color to the larvae [5,6]. For this reason, aquatic larvae of Chironomus are commonly called ‘blood worms’. Several unique properties of extracellular hemoglobin of Chironomids have been studied and reported in the literature. Total loss of introns could be seen in Hb gene in the insect Chironomus which attracted attention of scientists working on hemoglobin evolution [7]. Insects of genus Chironomus are widely distributed globally and can thrive under diverse and extreme environmental conditions [8e11]. Previous findings from our research group highlighted tolerance of Chironomid midges to very high dose (~3000 Gy) of gamma radiation [12e15]. This observation of the remarkable tolerance of chironomid midges to high doses of gamma radiation provided an impetus to look into the status of irradiated Hb of Chironomus and that of human since the threshold level of human Hb molecule to retain structural integrity is only 100 Gy or 1 Mrad [16]. In this paper, a detailed account of effect of gamma radiation on the structural integrity of ancient extracellular Hb of chironomid midge and intracellular Hb of human has been
372
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
presented. Having observed maintenance of structural integrity of Chironomus Hb at high threshold level of gamma radiation contrast to human Hb, bioinformatics approach was adopted to compare the amino acid compositions of the ‘heme binding pocket’ of Hb from human and the chironomid midge Chironomus thummi thummi. Our study revealed interesting findings which could explain the molecular basis of sensitivity of human Hb in the backdrop of evolutionary divergence. 2. Materials and methods 2.1. Rearing of Chironomus Mass rearing technique was used to grow cultures of Chironomus ramosus as described previously [17]. 2.1.1. Gamma radiation and hemoglobin preparations Gamma radiation exposure was given to all the samples using 60 Co source Gamma Cell (GC-5000) BRIT, Mumbai, with dose rate 52.6 Gy/min. Dosimetric studies and estimated lethal dose (LD) values for Chironomus larvae were carried out immediately after the gamma radiation exposure and during post irradiation recovery (PIR) period (refer Supplementary data). Two sub lethal doses of 1200 Gy (LD10) and 2400 Gy (LD50) were used for all the experiments and administered on both Chironomus and human Hb samples, categorized and abbreviated as follows: (a) ChHbwl: Chironomus hemoglobin extracted from irradiated whole larvae (b) ChHbhl: Chironomus hemoglobin extracted from irradiated hemolymph (c) ChHbcp: Size exclusion chromatography (SEC) purified Chironomus hemoglobin exposed to radiation. (d) HuHbpb: Hemoglobin extracted from irradiated human peripheral blood. (e) HuHbcp: Size exclusion chromatography (SEC) purified human hemoglobin (Sigma) exposed to radiation (details furnished in Supplementary information). Protein concentrations were measured using Bradford assay. 2.1.1.1. Chironomus hemoglobin samples. For ChHbwl, thirty day old fourth instar larvae were taken in a glass beaker containing water and exposed to gamma radiation along with unexposed control set. Larval hemolymph was collected using glass capillaries (inner diameter: 1 mm), centrifuged at 5000 rpm for 5 min, at 4 C. Supernatant containing extracellular Chironomus hemoglobin was used for further studies. Similar methodology was followed for ChHbhl samples. For ChHbcp, size exclusion chromatography (Superdex 10/300 G-75 GE, USA) was employed to obtain purified fraction of Chironomus Hb from the larval hemolymph. 2.1.1.2. Human hemoglobin samples. Human peripheral blood was irradiated followed by hemoglobin isolation. Five ml blood was collected in EDTA from healthy human volunteer with informed consent following institutional ethical guidelines [18]. Irradiated blood sample was mixed with equal volumes of lymphocyte separating medium (LSM) and centrifuged at 1500 rpm for 5 min at 4 C to separate plasma and RBCs. Ammonium chloride lysis solution containing NH4Cl, NaHCO3 and EDTA was added to lyse RBCs. Tubes were kept on ice for 15 min, supernatant containing Hb (HuHbpb) was obtained by centrifugation at 1500 rpm for 5 min at 4 C. Commercially available lyophilized human hemoglobin (H7379, Sigma Aldrich) was reconstituted freshly in buffer containing 50 mM Na-phosphate 100 mM NaCl buffer (pH 7.0) and
purified by SEC for HuHbcp. 2.1.2. Dynamic light scattering Hydrodynamic diameter of Chironomus Hb was measured in ZetasizerNano ZS (Malvern, UK). Stability of protein was maintained in buffer containing 50 mM Na -phosphate-100 mM saline buffer (pH7.0). The hydrodynamic diameter (Dh) of all samples were analyzed using software provided by Malvern. Sample concentration and volume used were 100 mg and 1 ml respectively in quartz cuvettes. 2.1.3. Ultra violet-visible (UV-VIS) absorbance spectroscopy Absorbance spectra of hemoglobin after gamma radiation exposure was recorded on UV-visible spectrophotometer (JASCO) at room temperature in the wavelength range of 300e600 nm using quartz cuvette of 1 cm path length. For absorbance spectrum buffered saline was considered as blank. 2.1.4. CDNN analysis of CD spectra Changes in secondary structure of Hb were measured using CD spectropolarimeter (JASCO J-815, Japan). CD spectra were recorded in near UV (190e250 nm) region with a scan speed of 20 nm/min and a band-width of 1.0 nm. Samples were taken in 1 mm quartz cuvette and average of three scans taken. The CD data were used to calculate secondary structure of Hb using CDNN 2.1 spectra analysis software with deconvolution program [19]. 2.1.5. Fluorescence spectroscopy The fluorescence spectra of Hb was taken after gamma exposure using spectrofluorimeter (JASCO, Japan) with 1.0 cm path length quartz cuvette. Hb solution was excited at 280 nm and 330 nm respectively and emission spectra was recorded in 300e700 nm range. The bandwidth of excitation and emission slit was set at 10 nm and scanning speed was 100 nm/min. 2.1.6. Bioinformatics analysis High resolution crystal structures of hemoglobin from Chironomus thummi thummi (CttHb) (PDB id: 1ECA; Resolution 1.4 Å) [20] and Human (PDB id: 2DN2, Resolution 2.1 Å) [21] were retrieved from RCSB PDB. To identify heme binding pocket residues of CttHb, Human hemoglobin (HuHbA and HuHbB) chains and intermolecular interaction, Ligand Explorer tool (using sphere of 4.0 A radius centered on heme group), LigPlot [22] and BioLip
Fig. 1. Dynamic light scattering measurements with increasing hydrodynamic diameter ± S. E. (d nm) of ChHbwl, ChHbhl, ChHbcp,, HuHbpb and HuHbcp after gamma radiation exposure. *Significant difference value P < 0.013 (MANOVA using SPPSS 8.0 with Tukey’s test).
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
373
Fig. 2. UV-visible spectra showing changes in soret peak after gamma radiation exposure. a. ChHbwl, b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp (Color figure online).
database was used [23,24]. These residues were confirmed based on the crystal structure data as well. A detailed comparison of the heme pocket lining residues from the three structures (CttHb, HuHbA, HuHbB) was carried out to investigate the heme retention by ChHb as observed in experimental data. The equivalence of the heme pocket lining amino acid residues of the three Hbs was substantiated based on pair-wise structural alignments of CttHb with HuHbA and HuHbB using DALI pair-wise alignment server. 3. Results 3.1. Dynamic light scattering For Chironomus Hb samples ChHbwl and ChHbhl, the changes in Dh values after irradiation (1200 Gy and 2400 Gy) were marginal when compared with control value 3.61 ± 0.07 nm. ChHbcp formed larger aggregates (Dh ¼ 6.24 ± 0.03 nm) compared to that of ChHbwl (5.08 ± 0.06 nm) after 2400 Gy dose of gamma radiation (Fig. 1). In case of HuHbpb, 1200 Gy of gamma irradiation exposure increased the Dh to 13.49 ± 0.18 nm from that of control 5.02 ± 0.11 nm as a result of formation of much larger aggregates of Hb molecules.
Following 2400 Gy irradiation HuHbpb showed four fold increase in Dh (19.41 nm ± 0.93 nm) and HuHbcp showed three fold increased in Dh (14.98 ± 0.24 nm) compared to respective control samples. 3.2. UV-visible spectroscopy Significant decrease in intensity of soret peak at 415 nm was observed in all ChHb samples after gamma radiation exposures (Fig. 2). Interestingly, although the peak intensity in irradiated samples of ChHbhl and ChHbcp (Fig. 2b and 2c) had declined compared to ChHbwl (Fig. 2a) there was no shift in soret position (near 415 nm) in all three Chironomus Hb samples. On the other hand, HuHbpb showed distinct shift of soret band (Fig. 2d) when irradiated at 1200 Gy and 2400 Gy. No such conformational alterations were observed in case of HuHbcp (Fig. 2e). 3.3. CD spectral analysis CD spectra of ChHbwl, ChHbhl and HuHbpb post gamma irradiation was used to analyze percentages of a/b helices using CDNN 2.1 program (Table 1). Control ChHbwl and ChHbhl showed 94.2 ± 4%
Table 1 CDNN analysis showing changes in contents of the a-helix and the b-sheets (parallel and anti-parallel) obtained by fitting CD spectra for Chironomus and Human Hb after gamma radiation exposure. Sample
Helix В-turn Random coils
ChHbwl
ChHbhl
ChHbcp
HuHbpb
HuHbcp
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
94.2 2.9 2.8
84.8 8.0 4.6
61.6 13.0 12.5
96.3 1.3 1.2
78.5 8.6 11.0
61.6 12.9 17.6
95.2 5.1 2.3
52.9 14.8 14.6
52.1 13.8 22.7
71.4 9.2 11.2
52.0 13.7 23.1
44.8 14.8 26.4
66.8 11.7 16.5
34.7 16.6 30.4
22.1 18.1 34.5
374
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Fig. 3. A: Fluorescence spectra after gamma radiation exposure of a. ChHbwl, b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp excited at 280 nm (Color figure online). B: Fluorescence spectra after gamma radiation exposure of a. ChHbwl (inlet showing expanded scale), b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp excited at 330 nm (Color figure online).
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378 Table 2 Composition of heme cavities for CttHb, HuHbA and HuHbB. CttHb
HuHbA
HuHbB
Ile34 Lys37 Phe38 His58 Arg61 Ile62 Phe65 Phe66 Phe83 His87 Arg90 Val92 Gln96 Leu97 Phe100
Tyr42 Phe43 His45 His58 Lys61 Val62* Leu83 Leu86 His87 Leu91 Val93 Asn97 Phe98 Leu101 Leu136
Phe41 Phe42 His63 Lys66 Val67 Ala70 Leu88 Leu91 His92 Leu96 Val98 Asn102 Phe103 Leu106 Leu141
*: Val62 in HuHbA was not included in the amino acid residues identified using Ligand Explorer or BioLip. However, it is documented as an important heme pocket residue in the description and analysis of the crystal structure of de-oxy state of human hemoglobin (Park et al. 2006).
and 96.3 ± 2% a-helix structure respectively, while control HuHbpb and HuHbcp showed more than 71.4 ± 4% and 66.8 ± 3 a-helical structure respectively. Higher percentage of a-helices in Chironomus Hb provided more resistivity. After 2400 Gy of gamma
375
irradiation, both ChHbwl and ChHbhl showed changes in a-helix percentage (61.6%) while for HuHbpb and HuHbcp percentage of helix content got changed to 44.8% and 22.1%. Conversion of a-helix into b-sheets due to unfolding of Hb secondary structure is irreversible, which contributed to damage of HuHb.
3.4. Fluorescence spectroscopy Fluorescence spectra of all irradiated samples showed reduced fluorescence intensity at 280 nm (Fig. 3A). 1200 Gy dose did not show any noticeable changes in ChHbwl,ChHbhl, and ChHbcp, while 2400 Gy dose showed two fold decline in fluorescence peak intensity for all 3 samples. Marked decrease in fluorescence intensity was observed in HuHbpb (~6 fold) and HuHbcp (~4 fold) samples when compared to ChHb. Fluorescence properties of porphyrin were analyzed to ascertain the molecular stability of ChHb and HuHb samples at the excitation wavelength of 330 nm. In irradiated samples loss of heme from porphyrin ring was evident from the increase in peak intensity at 440 nm emission spectra. For ChHbwl, the relative dissociation of iron was less compared to ChHbhl and ChHbcp (Fig. 3B). On the other hand, HuHbpb and HuHbcp showed approximately ~40 fold higher Fe dissociation compared to both experimental sets of ChHb i.e. ChHbwl and ChHbhl.
Fig. 4. Close-up views of the heme-binding pockets of (A) CttHb (front and back views), (B) HuHbA and (C) HuHbB. Amino acid residues lining the binding cavity are rendered as solid surfaces colored according to hydrophobicity. The heme groups are rendered as sticks. Rest of the hemoglobin structure is rendered as ribbons.
376
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Table 3 Comparison of structurally equivalent heme pocket lining amino acid residues* in CttHb, HuHbA and HuHbB. [*: Green cells: Hydrophobic Amino acids; Red cells: Hydrophilic amino acids] Position CttHb
HuHbA
HuHbB
Interaction with Heme
1
Ile34
Thr39
Thr38
2
Lys37
Tyr42
Phe41
3
Phe38
Phe43
Phe42
4
His58
His58
His63
Distal His
5
Arg61
Lys61
Lys66
Water-mediated H-bonds (2)
6
Ile62
Val62
Val67
7
Phe65
Ala65
Ala70
8
Phe66
Leu66
Phe71
9
Phe83
Leu83
Leu88
10
Ser86
Leu86
Leu91
No.
Water-mediated H-bond only in CttHb 11
His87
His87
His92
Proximal His
12
Arg90
Leu91
Leu96
Salt Bridge only in CttHb
13
Val92
Val93
Val98
14
Gln96
Asn97
Asn102
15
Leu97
Phe98
Phe103
16
Phe100
Leu101
Leu106
3.5. Bioinformatics analysis Comparative analysis of the heme binding pockets of CttHb with HuHbA and HuHbB was carried out to gain molecular level insights into the differential heme retention of these hemoglobins. As can be seen in Table 2 and Fig. 4, the amino acid compositions of the heme cavities of the three polypeptide chains differ from each other. Notable differences were seen with respect to Leu and Phe residues. The CttHb heme cavity is richer in Phe residues (5 out of 16) as compared to that of HuHbA and HuHbB (2 out of 18 and 3 out of 17). On the other hand, CttHb contains only one Leu while in HuHbA and HuHbB there are 5 and 6 Leu residues respectively. CttHb also has 2 Ile residues which are not seen in both the chains of human hemoglobin. Comparison of the 16 structurally equivalent amino acid residues of the three heme pockets (identified on the basis of structural alignment) revealed that, 4 amino acid residues out of 16 are conserved in all three (positions 3, 4, 11, 13 in Table 3). These conserved binding site positions include the proximal and distal His residues. Additionally, the conserved Phe and Val amino acid residues impart apolar nature to the heme pocket. Considering CttHb as a reference, the rest of the positions (12) have substitutions in either or both the human Hb chains. Among these; 3 contain physico-chemically similar amino acid residues (position numbers 5, 14: hydrophilic and position number 6: hydrophobic). With reference to CttHb, the remaining 9 variable positions show substitutions of the following types viz. (a) hydrophobic (aliphatic) to hydrophilic in position no. 1 (b) hydrophilic to hydrophobic aromatic/hydrophilic aromatic in position 2 (c)
hydrophobic aromatic to hydrophobic aliphatic in positions 7, 8, 9 and 16 (d) hydrophilic to hydrophobic aliphatic in positions 10, 12. (e) hydrophobic aliphatic to hydrophobic aromatic in position 15. This comparison indicated that, CttHb has more hydrophobic aromatic residues (Phe) interacting with heme group as compared to human hemoglobin A and B chains (which have more hydrophobic aliphatic residues). Three hydrophilic residues viz. Arg61, Ser86 and Arg90 in CttHb are involved in stabilizing electrostatic interactions with the heme group [23]. It may be noted from Table 2 that Ser86 and Arg90 of CttHb are substituted by hydrophobic amino acid residues in HuHbA and HuHbB implying the absence of the stabilizing electrostatic interactions. 4. Discussion This is the first comparative study on the stability of Chironomus and human Hb following gamma radiation exposure. Gamma radiation tolerance of Chironomus is 350 times higher than humans and our earlier studies [12e15] have found few of the many features of radiotolerant organism Chironomus ramosus. Present findings from biophysical and bioinformatics data further reinforce the fact from the perspective of hemoglobin present in this invertebrate. The hydrodynamic diameter obtained from DLS for all Chironomus Hb samples did not alter significantly post gamma irradiation. This analysis revealed higher level of aggregates and four fold increase in hydrodynamic diameter for human hemoglobins in a dose dependent manner (1200 Gye2400 Gy). Though unusual for human tolerance these
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
doses were selected for purpose of comparison only. Another criterion to measure damage of Hb molecule per se is the characteristic soret peak at 415 nm seen in UV-visible absorption spectra [25e27]. All irradiated ChHb samples showed decrease in intensity of soret peak with no change in the peak position. Irradiated human whole blood samples on the other hand shifted soret band towards longer wavelength due to conversion of Fe2þ e Fe3þ. This form of Fe has no physiological or functional relevance indicating 100% damage of HuHb. In CD spectroscopy study, ChHb showed more than 95% of alpha helical structure which was twofold higher than HuHb. Irradiation caused disruption of covalent bonds of the polypeptide chains of hemoglobin protein and transformed a-helical structure into bturns and random coils. Whether higher percentage of alpha helix confers radiation protection to ChHb would be an interesting area for further studies. Conformational change in proteins is reflected in their intrinsic fluorescence patterns. Tryptophan and tyrosine residues usually excite at 280 nm while for heme moieties in Hb molecule, effective quenching of fluorescence takes place when excited at 330 nm because of non covalent bond between iron and histidine [28]. Thus, we selected 280 nm and 330 nm excitation wavelengths to check protein degradation and release of iron from porphyrin ring of Hb respectively. Shifting of peak values indicated denaturation and quenching of heme in irradiated samples. Chironomus Hb samples revealed higher resistivity when subjected to gamma radiation relative to human Hb. All biochemical as well as biophysical data obtained in the present study indicated a higher degree of stability of ChHb post gamma irradiation and resistance to heme loss, hence we turned our attention to heme pockets. Further rationale for paying our attention to heme pocket got strengthened when fluorescence spectra targeted to 330 nm excitation wavelength provided a direct indication of heme loss and showed disturbed stability of Hb. Analyses of heme pockets of globins have been reported that correlate the sequence/structural features with the functions of globins [29]. The three dimensional structure of deoxy hemoglobin (component III) from Chironomus thummi thummi being the only reference structure available for any globin from this genus, it was used for comparative analysis. Moreover, the biochemical similarities of Hb components of Chironomus thummi thummi and C. ramosus have been shown in earlier studies by Das and Handique [30]. In the light of the observed differences of Chironomus and human hemoglobins with respect to resistance to gamma rays, sequence and structure-based bioinformatics analyses were carried out with an objective to assess any correlations of the sequence/ structural properties with the heme retention ability. Hemebinding pockets of CttHb, HuHbA and HuHbB highlight the differences in the amino acid composition and the interactions between each of the globins and the respective heme groups. Dominance of aromatic hydrophobic amino acids (Phe) and characteristic electrostatic interactions of Ser86 and Arg90 in CttHb are the two major features that emerged from this analysis and are likely to be conferring better heme retention ability to CttHb. Previous studies had also shown that the stability of porphyrin ring in hemoglobin was attributed to Phe residues of the polypeptide chain [29]. Disruption of the hydrophobic interactions of these residues severely affects heme retention in the binding pocket of hemoglobin and leads to heme loss in case of HuHb. These observations complemented our empirical data of comparative analysis of Chironomus and human hemoglobin and provide an explanation for the vulnerability of human hemoglobin molecule to high dose of gamma radiation. Gamma radiation tolerance of Chironomid midges might have resulted from better ability of heme retention of the ancient hemoglobin protein during the course of evolution.
377
Acknowledgement Authors acknowledge use of all the facilities provided in Molecular Biology Division, Solid State Physics Division of the Bhabha Atomic Research Centre, Mumbai. Support from Department of Atomic Energy (DAE) India, for fellowship to PG under Bhabha Atomic Research Centre- Savitribai Phule Pune University (BARCSPPU: GoI-E-159) collaborative PhD programme (BBN and RM) is duly accredited. BBN acknowledges partial support received from SPPU under University Grant Commission- Centre for Advance Studies (UGC-CAS: II-255) and Department of Science and Technology- Promotion of University Research and Scientific Excellence (DST-PURSE: GoI-A670) grants. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.05.129. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.129. References [1] R. Hardison, Hemoglobins from bacteria to man: evolution of different patterns of gene expression, J. Exp. Biol. 201 (1998) 1099e1117. [2] S.N. Vinogradov, D.A. Walz, B. Pohajdak, L. Moens, O.H. Kapp, T. Suzuki, C.N. Trotman, Adventitious variability? the amino acid sequences of nonvertebrate globins, Comp. Biochem. Physiol. B 106 (1993) 1e26. [3] R.E. Weber, S.N. Vinogradov, Non vertebrate hemoglobins: functions and molecular adaptations, Physiol. Rev. 81 (2001) 569e628. [4] S.N. Vinogradov, D. Hoogewijs, X. Bailly, R. Arredondo-Peter, M. Guertin, J. Gough, S. Dewilde, L. Moens, J.R. Vanfleteren, Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life, Prof. Natl. Acad. Sci. 102 (2005) 11385e11389. [5] P. Armitage, P.S. Cranston, L.C.V. Pinder (Eds.), Chironomidae: Biology and Ecology of Non-biting Midges, Chapman and Hall, London, 1995. [6] T. Burmester, T. Hankeln, The respiratory proteins of insects, J. Insect Physiol. 53 (2007) 285e294. [7] M. Antoine, J. Niessing, Intron-less globin genes in the insect Chironomus thummi, Nature 310 (1984) 795e798. [8] B. Nagell, C.C. Landahl, Resistance to anoxia of Chironomus plumosus and Chironomus anthracinus (Diptera) larvae, Holarct. Ecol. 1 (1978) 333e336. [9] L.C.V. Pinder, Biology of freshwater Chironomidae, Annu. Rev. Entomol. 37 (1986) 1e23. [10] D.R. Oliver, Life history of Chironomidae, Annu. Rev. Entomol. 16 (1971) 211e230. [11] L.C. Ferrington, Global diversity of non-biting midges (Chironomidae; Insecta e Diptera) in freshwater, Hydrobiologia 595 (2008) 447e455. [12] K.D. Datkhile, T.K. Dongre, R. Mukhopadhyaya, B.B. Nath, Gamma radiation tolerance of a tropical species of midge Chironomus ramosus Chaudhuri (Diptera: Chironomidae), Int. J. Radiat. Biol. 85 (2009a) 1e9. [13] K.D. Datkhile, R. Mukhopadhyaya, T.K. Dongre, B.B. Nath, Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation, Comp. Biochem. Physiol. Part C. Pharmacol. Toxicol. Endocrinol. 149 (2009b) 500e506. [14] K.D. Datkhile, R. Mukhopadhyaya, T.K. Dongre, B.B. Nath, Hsp70 expression in Chironomus ramosus exposed to gamma radiation, Int. J. Radiat. Biol. 87 (2011) 213e221. [15] K.D. Datkhile, P.S. Gaikwad, R. Mukhopadhyaya, S.S. Ghaskadbi, B.B. Nath, Chironomus ramosus larvae exhibit DNA damage control in response to gamma radiation, Int. J. Radiat. Biol. 91 (2015) 742e748. [16] Z. Szweda-Lewandowska, M. Puchola, W. Leyko, Effects of gamma irradiation on the structure and function of human hemoglobin, Radiat. Res. 65 (1976) 50e59. [17] B.B. Nath, N.N. Godbole, Technique for mass rearing of Indian Chironomus species (Diptera, Nematocera, Chironomidae), Stud. Dipterol. 5 (1998) 187e193. [18] A. Kumar, M. Ali, R.S. Ningthoujam, P. Gaikwad, M. Kumar, B.B. Nath, B.N. Pandey, The interaction of actinide and lanthanide ions with hemoglobin and its relevance to human and environmental toxicology, J. Hazard. Mater. 307 (2015) 281e293. [19] G. Ghosh, L. Panicker, R.S. Ningthoujam, K.C. Barick, R. Tewari, Counter ion induced irreversible denaturation of hen egg white lysozyme upon electrostatic interaction with iron oxide nanoparticles: a predicted model, Colloid
378
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Surf. B Biointer. (2013) 103267. [20] J. Yang, A. Roy, Y. Zhang, BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions, Nucleic Acids Res. 41 (2013) D1096eD1103. [21] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions, Protein Eng. 8 (1996) 127e134. [22] J.L. Moreland, A. Gramada, O.V. Buzko, Q. Zhang, P.E. Bourne, The Molecular Biology Toolkit (MBT): a modular platform for developing molecular visualization applications, BMC Bioinforma. 6 (2005) 21. [23] W. Steigemann, E. Weber, Structure of erythrocruorin in different ligand states refined at 1.4 A resolution, J. Mol. Biol. 127 (1979) 309e338. [24] S.Y. Park, T. Yokoyama, N. Shibayama, Y. Shiro, J.R.H. Tame, 1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms, J. Mol. Biol. 360 (2006) 690e701. [25] W.G. Zijlstra, A. Buursma, W.P. Meeuwsen-van der Roest, Absorption spectra
[26]
[27] [28] [29]
[30]
of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin, Clin. Chem. 37 (1991) 1633. D.J. Faber, M.C.G. Aalders, E.G. Mik, B.A. Hooper, M.J.C. van Gemert, T.G. van Leeuwen, Oxygen saturation-dependent absorption and scattering of blood, Phys. Rev. Lett. 93 (2004) 028102. L. Gebicka, E. Banasiak, Flavonoids as reductants of ferryl haemoglobin, Acta Biochim. Pol. 56 (2009) 509e513. N. Ramajunam, K. Vishwanathan, Fluorescence spectroscopy in vivo, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, 2011, pp. 20e56. E.C. Liong, Y. Dou, E.E. Scott, J.S. Olson, G.N. Phillips Jr., Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin, J. Biochem. 276 (2001) 9093e9100. R. Das, R. Handique, Hemoglobin in Chironomus ramosus (Insecta, Diptera): an electrophoretic study of polymorphism, developmental sequence and interspecific relationship, Hydrobiologia 318 (1996a) 43e50.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Differential sensitivity of Chironomus and human hemoglobin to gamma radiation Pallavi S. Gaikwad a, b, Lata Panicker c, Madhura Mohole d, Sangeeta Sawant d, Rita Mukhopadhyaya b, Bimalendu B. Nath a, * a
Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule University, Pune, 411007, India Molecular Biology Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai, 400085, India d Bioinformatics Center, Savitribai Phule Pune University, Pune, 411007, India b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 May 2016 Accepted 25 May 2016 Available online 26 May 2016
Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. © 2016 Elsevier Inc. All rights reserved.
Keywords: Chironomus hemoglobin Gamma radiation tolerance Human hemoglobin Heme-binding pocket analysis
1. Introduction Hemoglobin (Hb) constitutes a remarkable family of genes and proteins present across all phyla. It has a long history of 600e800 million years of evolution [1]. Hemoglobins (Hbs) are intracellular in jawed vertebrates, found exclusively in erythroid cells in sharp contrast to rarely encountered invertebrate animals where Hbs are extracellular [2,3]. In adult human, Hb is a heterotetramer, consisting of two a-globins and two b-globin polypeptides conjugated with their heme groups [4]. Down the evolutionary line, one can encounter an ancient group of dipteran insects, the chironomid midges that evolved approximately 200 million years ago and are the only insects possessing Hb in the hemolymph conferring
* Corresponding author. Stress Biology Research Laboratory, Department of Zoology, Savitribai Phule Pune University, Pune, 411 007, India. E-mail addresses: [email protected], [email protected] (B.B. Nath). http://dx.doi.org/10.1016/j.bbrc.2016.05.129 0006-291X/© 2016 Elsevier Inc. All rights reserved.
characteristic red color to the larvae [5,6]. For this reason, aquatic larvae of Chironomus are commonly called ‘blood worms’. Several unique properties of extracellular hemoglobin of Chironomids have been studied and reported in the literature. Total loss of introns could be seen in Hb gene in the insect Chironomus which attracted attention of scientists working on hemoglobin evolution [7]. Insects of genus Chironomus are widely distributed globally and can thrive under diverse and extreme environmental conditions [8e11]. Previous findings from our research group highlighted tolerance of Chironomid midges to very high dose (~3000 Gy) of gamma radiation [12e15]. This observation of the remarkable tolerance of chironomid midges to high doses of gamma radiation provided an impetus to look into the status of irradiated Hb of Chironomus and that of human since the threshold level of human Hb molecule to retain structural integrity is only 100 Gy or 1 Mrad [16]. In this paper, a detailed account of effect of gamma radiation on the structural integrity of ancient extracellular Hb of chironomid midge and intracellular Hb of human has been
372
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
presented. Having observed maintenance of structural integrity of Chironomus Hb at high threshold level of gamma radiation contrast to human Hb, bioinformatics approach was adopted to compare the amino acid compositions of the ‘heme binding pocket’ of Hb from human and the chironomid midge Chironomus thummi thummi. Our study revealed interesting findings which could explain the molecular basis of sensitivity of human Hb in the backdrop of evolutionary divergence. 2. Materials and methods 2.1. Rearing of Chironomus Mass rearing technique was used to grow cultures of Chironomus ramosus as described previously [17]. 2.1.1. Gamma radiation and hemoglobin preparations Gamma radiation exposure was given to all the samples using 60 Co source Gamma Cell (GC-5000) BRIT, Mumbai, with dose rate 52.6 Gy/min. Dosimetric studies and estimated lethal dose (LD) values for Chironomus larvae were carried out immediately after the gamma radiation exposure and during post irradiation recovery (PIR) period (refer Supplementary data). Two sub lethal doses of 1200 Gy (LD10) and 2400 Gy (LD50) were used for all the experiments and administered on both Chironomus and human Hb samples, categorized and abbreviated as follows: (a) ChHbwl: Chironomus hemoglobin extracted from irradiated whole larvae (b) ChHbhl: Chironomus hemoglobin extracted from irradiated hemolymph (c) ChHbcp: Size exclusion chromatography (SEC) purified Chironomus hemoglobin exposed to radiation. (d) HuHbpb: Hemoglobin extracted from irradiated human peripheral blood. (e) HuHbcp: Size exclusion chromatography (SEC) purified human hemoglobin (Sigma) exposed to radiation (details furnished in Supplementary information). Protein concentrations were measured using Bradford assay. 2.1.1.1. Chironomus hemoglobin samples. For ChHbwl, thirty day old fourth instar larvae were taken in a glass beaker containing water and exposed to gamma radiation along with unexposed control set. Larval hemolymph was collected using glass capillaries (inner diameter: 1 mm), centrifuged at 5000 rpm for 5 min, at 4 C. Supernatant containing extracellular Chironomus hemoglobin was used for further studies. Similar methodology was followed for ChHbhl samples. For ChHbcp, size exclusion chromatography (Superdex 10/300 G-75 GE, USA) was employed to obtain purified fraction of Chironomus Hb from the larval hemolymph. 2.1.1.2. Human hemoglobin samples. Human peripheral blood was irradiated followed by hemoglobin isolation. Five ml blood was collected in EDTA from healthy human volunteer with informed consent following institutional ethical guidelines [18]. Irradiated blood sample was mixed with equal volumes of lymphocyte separating medium (LSM) and centrifuged at 1500 rpm for 5 min at 4 C to separate plasma and RBCs. Ammonium chloride lysis solution containing NH4Cl, NaHCO3 and EDTA was added to lyse RBCs. Tubes were kept on ice for 15 min, supernatant containing Hb (HuHbpb) was obtained by centrifugation at 1500 rpm for 5 min at 4 C. Commercially available lyophilized human hemoglobin (H7379, Sigma Aldrich) was reconstituted freshly in buffer containing 50 mM Na-phosphate 100 mM NaCl buffer (pH 7.0) and
purified by SEC for HuHbcp. 2.1.2. Dynamic light scattering Hydrodynamic diameter of Chironomus Hb was measured in ZetasizerNano ZS (Malvern, UK). Stability of protein was maintained in buffer containing 50 mM Na -phosphate-100 mM saline buffer (pH7.0). The hydrodynamic diameter (Dh) of all samples were analyzed using software provided by Malvern. Sample concentration and volume used were 100 mg and 1 ml respectively in quartz cuvettes. 2.1.3. Ultra violet-visible (UV-VIS) absorbance spectroscopy Absorbance spectra of hemoglobin after gamma radiation exposure was recorded on UV-visible spectrophotometer (JASCO) at room temperature in the wavelength range of 300e600 nm using quartz cuvette of 1 cm path length. For absorbance spectrum buffered saline was considered as blank. 2.1.4. CDNN analysis of CD spectra Changes in secondary structure of Hb were measured using CD spectropolarimeter (JASCO J-815, Japan). CD spectra were recorded in near UV (190e250 nm) region with a scan speed of 20 nm/min and a band-width of 1.0 nm. Samples were taken in 1 mm quartz cuvette and average of three scans taken. The CD data were used to calculate secondary structure of Hb using CDNN 2.1 spectra analysis software with deconvolution program [19]. 2.1.5. Fluorescence spectroscopy The fluorescence spectra of Hb was taken after gamma exposure using spectrofluorimeter (JASCO, Japan) with 1.0 cm path length quartz cuvette. Hb solution was excited at 280 nm and 330 nm respectively and emission spectra was recorded in 300e700 nm range. The bandwidth of excitation and emission slit was set at 10 nm and scanning speed was 100 nm/min. 2.1.6. Bioinformatics analysis High resolution crystal structures of hemoglobin from Chironomus thummi thummi (CttHb) (PDB id: 1ECA; Resolution 1.4 Å) [20] and Human (PDB id: 2DN2, Resolution 2.1 Å) [21] were retrieved from RCSB PDB. To identify heme binding pocket residues of CttHb, Human hemoglobin (HuHbA and HuHbB) chains and intermolecular interaction, Ligand Explorer tool (using sphere of 4.0 A radius centered on heme group), LigPlot [22] and BioLip
Fig. 1. Dynamic light scattering measurements with increasing hydrodynamic diameter ± S. E. (d nm) of ChHbwl, ChHbhl, ChHbcp,, HuHbpb and HuHbcp after gamma radiation exposure. *Significant difference value P < 0.013 (MANOVA using SPPSS 8.0 with Tukey’s test).
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
373
Fig. 2. UV-visible spectra showing changes in soret peak after gamma radiation exposure. a. ChHbwl, b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp (Color figure online).
database was used [23,24]. These residues were confirmed based on the crystal structure data as well. A detailed comparison of the heme pocket lining residues from the three structures (CttHb, HuHbA, HuHbB) was carried out to investigate the heme retention by ChHb as observed in experimental data. The equivalence of the heme pocket lining amino acid residues of the three Hbs was substantiated based on pair-wise structural alignments of CttHb with HuHbA and HuHbB using DALI pair-wise alignment server. 3. Results 3.1. Dynamic light scattering For Chironomus Hb samples ChHbwl and ChHbhl, the changes in Dh values after irradiation (1200 Gy and 2400 Gy) were marginal when compared with control value 3.61 ± 0.07 nm. ChHbcp formed larger aggregates (Dh ¼ 6.24 ± 0.03 nm) compared to that of ChHbwl (5.08 ± 0.06 nm) after 2400 Gy dose of gamma radiation (Fig. 1). In case of HuHbpb, 1200 Gy of gamma irradiation exposure increased the Dh to 13.49 ± 0.18 nm from that of control 5.02 ± 0.11 nm as a result of formation of much larger aggregates of Hb molecules.
Following 2400 Gy irradiation HuHbpb showed four fold increase in Dh (19.41 nm ± 0.93 nm) and HuHbcp showed three fold increased in Dh (14.98 ± 0.24 nm) compared to respective control samples. 3.2. UV-visible spectroscopy Significant decrease in intensity of soret peak at 415 nm was observed in all ChHb samples after gamma radiation exposures (Fig. 2). Interestingly, although the peak intensity in irradiated samples of ChHbhl and ChHbcp (Fig. 2b and 2c) had declined compared to ChHbwl (Fig. 2a) there was no shift in soret position (near 415 nm) in all three Chironomus Hb samples. On the other hand, HuHbpb showed distinct shift of soret band (Fig. 2d) when irradiated at 1200 Gy and 2400 Gy. No such conformational alterations were observed in case of HuHbcp (Fig. 2e). 3.3. CD spectral analysis CD spectra of ChHbwl, ChHbhl and HuHbpb post gamma irradiation was used to analyze percentages of a/b helices using CDNN 2.1 program (Table 1). Control ChHbwl and ChHbhl showed 94.2 ± 4%
Table 1 CDNN analysis showing changes in contents of the a-helix and the b-sheets (parallel and anti-parallel) obtained by fitting CD spectra for Chironomus and Human Hb after gamma radiation exposure. Sample
Helix В-turn Random coils
ChHbwl
ChHbhl
ChHbcp
HuHbpb
HuHbcp
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
Control
1200 Gy
2400 Gy
94.2 2.9 2.8
84.8 8.0 4.6
61.6 13.0 12.5
96.3 1.3 1.2
78.5 8.6 11.0
61.6 12.9 17.6
95.2 5.1 2.3
52.9 14.8 14.6
52.1 13.8 22.7
71.4 9.2 11.2
52.0 13.7 23.1
44.8 14.8 26.4
66.8 11.7 16.5
34.7 16.6 30.4
22.1 18.1 34.5
374
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Fig. 3. A: Fluorescence spectra after gamma radiation exposure of a. ChHbwl, b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp excited at 280 nm (Color figure online). B: Fluorescence spectra after gamma radiation exposure of a. ChHbwl (inlet showing expanded scale), b. ChHbhl, c. ChHbcp, d. HuHbpb and e. HuHbcp excited at 330 nm (Color figure online).
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378 Table 2 Composition of heme cavities for CttHb, HuHbA and HuHbB. CttHb
HuHbA
HuHbB
Ile34 Lys37 Phe38 His58 Arg61 Ile62 Phe65 Phe66 Phe83 His87 Arg90 Val92 Gln96 Leu97 Phe100
Tyr42 Phe43 His45 His58 Lys61 Val62* Leu83 Leu86 His87 Leu91 Val93 Asn97 Phe98 Leu101 Leu136
Phe41 Phe42 His63 Lys66 Val67 Ala70 Leu88 Leu91 His92 Leu96 Val98 Asn102 Phe103 Leu106 Leu141
*: Val62 in HuHbA was not included in the amino acid residues identified using Ligand Explorer or BioLip. However, it is documented as an important heme pocket residue in the description and analysis of the crystal structure of de-oxy state of human hemoglobin (Park et al. 2006).
and 96.3 ± 2% a-helix structure respectively, while control HuHbpb and HuHbcp showed more than 71.4 ± 4% and 66.8 ± 3 a-helical structure respectively. Higher percentage of a-helices in Chironomus Hb provided more resistivity. After 2400 Gy of gamma
375
irradiation, both ChHbwl and ChHbhl showed changes in a-helix percentage (61.6%) while for HuHbpb and HuHbcp percentage of helix content got changed to 44.8% and 22.1%. Conversion of a-helix into b-sheets due to unfolding of Hb secondary structure is irreversible, which contributed to damage of HuHb.
3.4. Fluorescence spectroscopy Fluorescence spectra of all irradiated samples showed reduced fluorescence intensity at 280 nm (Fig. 3A). 1200 Gy dose did not show any noticeable changes in ChHbwl,ChHbhl, and ChHbcp, while 2400 Gy dose showed two fold decline in fluorescence peak intensity for all 3 samples. Marked decrease in fluorescence intensity was observed in HuHbpb (~6 fold) and HuHbcp (~4 fold) samples when compared to ChHb. Fluorescence properties of porphyrin were analyzed to ascertain the molecular stability of ChHb and HuHb samples at the excitation wavelength of 330 nm. In irradiated samples loss of heme from porphyrin ring was evident from the increase in peak intensity at 440 nm emission spectra. For ChHbwl, the relative dissociation of iron was less compared to ChHbhl and ChHbcp (Fig. 3B). On the other hand, HuHbpb and HuHbcp showed approximately ~40 fold higher Fe dissociation compared to both experimental sets of ChHb i.e. ChHbwl and ChHbhl.
Fig. 4. Close-up views of the heme-binding pockets of (A) CttHb (front and back views), (B) HuHbA and (C) HuHbB. Amino acid residues lining the binding cavity are rendered as solid surfaces colored according to hydrophobicity. The heme groups are rendered as sticks. Rest of the hemoglobin structure is rendered as ribbons.
376
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Table 3 Comparison of structurally equivalent heme pocket lining amino acid residues* in CttHb, HuHbA and HuHbB. [*: Green cells: Hydrophobic Amino acids; Red cells: Hydrophilic amino acids] Position CttHb
HuHbA
HuHbB
Interaction with Heme
1
Ile34
Thr39
Thr38
2
Lys37
Tyr42
Phe41
3
Phe38
Phe43
Phe42
4
His58
His58
His63
Distal His
5
Arg61
Lys61
Lys66
Water-mediated H-bonds (2)
6
Ile62
Val62
Val67
7
Phe65
Ala65
Ala70
8
Phe66
Leu66
Phe71
9
Phe83
Leu83
Leu88
10
Ser86
Leu86
Leu91
No.
Water-mediated H-bond only in CttHb 11
His87
His87
His92
Proximal His
12
Arg90
Leu91
Leu96
Salt Bridge only in CttHb
13
Val92
Val93
Val98
14
Gln96
Asn97
Asn102
15
Leu97
Phe98
Phe103
16
Phe100
Leu101
Leu106
3.5. Bioinformatics analysis Comparative analysis of the heme binding pockets of CttHb with HuHbA and HuHbB was carried out to gain molecular level insights into the differential heme retention of these hemoglobins. As can be seen in Table 2 and Fig. 4, the amino acid compositions of the heme cavities of the three polypeptide chains differ from each other. Notable differences were seen with respect to Leu and Phe residues. The CttHb heme cavity is richer in Phe residues (5 out of 16) as compared to that of HuHbA and HuHbB (2 out of 18 and 3 out of 17). On the other hand, CttHb contains only one Leu while in HuHbA and HuHbB there are 5 and 6 Leu residues respectively. CttHb also has 2 Ile residues which are not seen in both the chains of human hemoglobin. Comparison of the 16 structurally equivalent amino acid residues of the three heme pockets (identified on the basis of structural alignment) revealed that, 4 amino acid residues out of 16 are conserved in all three (positions 3, 4, 11, 13 in Table 3). These conserved binding site positions include the proximal and distal His residues. Additionally, the conserved Phe and Val amino acid residues impart apolar nature to the heme pocket. Considering CttHb as a reference, the rest of the positions (12) have substitutions in either or both the human Hb chains. Among these; 3 contain physico-chemically similar amino acid residues (position numbers 5, 14: hydrophilic and position number 6: hydrophobic). With reference to CttHb, the remaining 9 variable positions show substitutions of the following types viz. (a) hydrophobic (aliphatic) to hydrophilic in position no. 1 (b) hydrophilic to hydrophobic aromatic/hydrophilic aromatic in position 2 (c)
hydrophobic aromatic to hydrophobic aliphatic in positions 7, 8, 9 and 16 (d) hydrophilic to hydrophobic aliphatic in positions 10, 12. (e) hydrophobic aliphatic to hydrophobic aromatic in position 15. This comparison indicated that, CttHb has more hydrophobic aromatic residues (Phe) interacting with heme group as compared to human hemoglobin A and B chains (which have more hydrophobic aliphatic residues). Three hydrophilic residues viz. Arg61, Ser86 and Arg90 in CttHb are involved in stabilizing electrostatic interactions with the heme group [23]. It may be noted from Table 2 that Ser86 and Arg90 of CttHb are substituted by hydrophobic amino acid residues in HuHbA and HuHbB implying the absence of the stabilizing electrostatic interactions. 4. Discussion This is the first comparative study on the stability of Chironomus and human Hb following gamma radiation exposure. Gamma radiation tolerance of Chironomus is 350 times higher than humans and our earlier studies [12e15] have found few of the many features of radiotolerant organism Chironomus ramosus. Present findings from biophysical and bioinformatics data further reinforce the fact from the perspective of hemoglobin present in this invertebrate. The hydrodynamic diameter obtained from DLS for all Chironomus Hb samples did not alter significantly post gamma irradiation. This analysis revealed higher level of aggregates and four fold increase in hydrodynamic diameter for human hemoglobins in a dose dependent manner (1200 Gye2400 Gy). Though unusual for human tolerance these
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
doses were selected for purpose of comparison only. Another criterion to measure damage of Hb molecule per se is the characteristic soret peak at 415 nm seen in UV-visible absorption spectra [25e27]. All irradiated ChHb samples showed decrease in intensity of soret peak with no change in the peak position. Irradiated human whole blood samples on the other hand shifted soret band towards longer wavelength due to conversion of Fe2þ e Fe3þ. This form of Fe has no physiological or functional relevance indicating 100% damage of HuHb. In CD spectroscopy study, ChHb showed more than 95% of alpha helical structure which was twofold higher than HuHb. Irradiation caused disruption of covalent bonds of the polypeptide chains of hemoglobin protein and transformed a-helical structure into bturns and random coils. Whether higher percentage of alpha helix confers radiation protection to ChHb would be an interesting area for further studies. Conformational change in proteins is reflected in their intrinsic fluorescence patterns. Tryptophan and tyrosine residues usually excite at 280 nm while for heme moieties in Hb molecule, effective quenching of fluorescence takes place when excited at 330 nm because of non covalent bond between iron and histidine [28]. Thus, we selected 280 nm and 330 nm excitation wavelengths to check protein degradation and release of iron from porphyrin ring of Hb respectively. Shifting of peak values indicated denaturation and quenching of heme in irradiated samples. Chironomus Hb samples revealed higher resistivity when subjected to gamma radiation relative to human Hb. All biochemical as well as biophysical data obtained in the present study indicated a higher degree of stability of ChHb post gamma irradiation and resistance to heme loss, hence we turned our attention to heme pockets. Further rationale for paying our attention to heme pocket got strengthened when fluorescence spectra targeted to 330 nm excitation wavelength provided a direct indication of heme loss and showed disturbed stability of Hb. Analyses of heme pockets of globins have been reported that correlate the sequence/structural features with the functions of globins [29]. The three dimensional structure of deoxy hemoglobin (component III) from Chironomus thummi thummi being the only reference structure available for any globin from this genus, it was used for comparative analysis. Moreover, the biochemical similarities of Hb components of Chironomus thummi thummi and C. ramosus have been shown in earlier studies by Das and Handique [30]. In the light of the observed differences of Chironomus and human hemoglobins with respect to resistance to gamma rays, sequence and structure-based bioinformatics analyses were carried out with an objective to assess any correlations of the sequence/ structural properties with the heme retention ability. Hemebinding pockets of CttHb, HuHbA and HuHbB highlight the differences in the amino acid composition and the interactions between each of the globins and the respective heme groups. Dominance of aromatic hydrophobic amino acids (Phe) and characteristic electrostatic interactions of Ser86 and Arg90 in CttHb are the two major features that emerged from this analysis and are likely to be conferring better heme retention ability to CttHb. Previous studies had also shown that the stability of porphyrin ring in hemoglobin was attributed to Phe residues of the polypeptide chain [29]. Disruption of the hydrophobic interactions of these residues severely affects heme retention in the binding pocket of hemoglobin and leads to heme loss in case of HuHb. These observations complemented our empirical data of comparative analysis of Chironomus and human hemoglobin and provide an explanation for the vulnerability of human hemoglobin molecule to high dose of gamma radiation. Gamma radiation tolerance of Chironomid midges might have resulted from better ability of heme retention of the ancient hemoglobin protein during the course of evolution.
377
Acknowledgement Authors acknowledge use of all the facilities provided in Molecular Biology Division, Solid State Physics Division of the Bhabha Atomic Research Centre, Mumbai. Support from Department of Atomic Energy (DAE) India, for fellowship to PG under Bhabha Atomic Research Centre- Savitribai Phule Pune University (BARCSPPU: GoI-E-159) collaborative PhD programme (BBN and RM) is duly accredited. BBN acknowledges partial support received from SPPU under University Grant Commission- Centre for Advance Studies (UGC-CAS: II-255) and Department of Science and Technology- Promotion of University Research and Scientific Excellence (DST-PURSE: GoI-A670) grants. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.05.129. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.05.129. References [1] R. Hardison, Hemoglobins from bacteria to man: evolution of different patterns of gene expression, J. Exp. Biol. 201 (1998) 1099e1117. [2] S.N. Vinogradov, D.A. Walz, B. Pohajdak, L. Moens, O.H. Kapp, T. Suzuki, C.N. Trotman, Adventitious variability? the amino acid sequences of nonvertebrate globins, Comp. Biochem. Physiol. B 106 (1993) 1e26. [3] R.E. Weber, S.N. Vinogradov, Non vertebrate hemoglobins: functions and molecular adaptations, Physiol. Rev. 81 (2001) 569e628. [4] S.N. Vinogradov, D. Hoogewijs, X. Bailly, R. Arredondo-Peter, M. Guertin, J. Gough, S. Dewilde, L. Moens, J.R. Vanfleteren, Three globin lineages belonging to two structural classes in genomes from the three kingdoms of life, Prof. Natl. Acad. Sci. 102 (2005) 11385e11389. [5] P. Armitage, P.S. Cranston, L.C.V. Pinder (Eds.), Chironomidae: Biology and Ecology of Non-biting Midges, Chapman and Hall, London, 1995. [6] T. Burmester, T. Hankeln, The respiratory proteins of insects, J. Insect Physiol. 53 (2007) 285e294. [7] M. Antoine, J. Niessing, Intron-less globin genes in the insect Chironomus thummi, Nature 310 (1984) 795e798. [8] B. Nagell, C.C. Landahl, Resistance to anoxia of Chironomus plumosus and Chironomus anthracinus (Diptera) larvae, Holarct. Ecol. 1 (1978) 333e336. [9] L.C.V. Pinder, Biology of freshwater Chironomidae, Annu. Rev. Entomol. 37 (1986) 1e23. [10] D.R. Oliver, Life history of Chironomidae, Annu. Rev. Entomol. 16 (1971) 211e230. [11] L.C. Ferrington, Global diversity of non-biting midges (Chironomidae; Insecta e Diptera) in freshwater, Hydrobiologia 595 (2008) 447e455. [12] K.D. Datkhile, T.K. Dongre, R. Mukhopadhyaya, B.B. Nath, Gamma radiation tolerance of a tropical species of midge Chironomus ramosus Chaudhuri (Diptera: Chironomidae), Int. J. Radiat. Biol. 85 (2009a) 1e9. [13] K.D. Datkhile, R. Mukhopadhyaya, T.K. Dongre, B.B. Nath, Increased level of superoxide dismutase (SOD) activity in larvae of Chironomus ramosus (Diptera: Chironomidae) subjected to ionizing radiation, Comp. Biochem. Physiol. Part C. Pharmacol. Toxicol. Endocrinol. 149 (2009b) 500e506. [14] K.D. Datkhile, R. Mukhopadhyaya, T.K. Dongre, B.B. Nath, Hsp70 expression in Chironomus ramosus exposed to gamma radiation, Int. J. Radiat. Biol. 87 (2011) 213e221. [15] K.D. Datkhile, P.S. Gaikwad, R. Mukhopadhyaya, S.S. Ghaskadbi, B.B. Nath, Chironomus ramosus larvae exhibit DNA damage control in response to gamma radiation, Int. J. Radiat. Biol. 91 (2015) 742e748. [16] Z. Szweda-Lewandowska, M. Puchola, W. Leyko, Effects of gamma irradiation on the structure and function of human hemoglobin, Radiat. Res. 65 (1976) 50e59. [17] B.B. Nath, N.N. Godbole, Technique for mass rearing of Indian Chironomus species (Diptera, Nematocera, Chironomidae), Stud. Dipterol. 5 (1998) 187e193. [18] A. Kumar, M. Ali, R.S. Ningthoujam, P. Gaikwad, M. Kumar, B.B. Nath, B.N. Pandey, The interaction of actinide and lanthanide ions with hemoglobin and its relevance to human and environmental toxicology, J. Hazard. Mater. 307 (2015) 281e293. [19] G. Ghosh, L. Panicker, R.S. Ningthoujam, K.C. Barick, R. Tewari, Counter ion induced irreversible denaturation of hen egg white lysozyme upon electrostatic interaction with iron oxide nanoparticles: a predicted model, Colloid
378
P.S. Gaikwad et al. / Biochemical and Biophysical Research Communications 476 (2016) 371e378
Surf. B Biointer. (2013) 103267. [20] J. Yang, A. Roy, Y. Zhang, BioLiP: a semi-manually curated database for biologically relevant ligand-protein interactions, Nucleic Acids Res. 41 (2013) D1096eD1103. [21] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions, Protein Eng. 8 (1996) 127e134. [22] J.L. Moreland, A. Gramada, O.V. Buzko, Q. Zhang, P.E. Bourne, The Molecular Biology Toolkit (MBT): a modular platform for developing molecular visualization applications, BMC Bioinforma. 6 (2005) 21. [23] W. Steigemann, E. Weber, Structure of erythrocruorin in different ligand states refined at 1.4 A resolution, J. Mol. Biol. 127 (1979) 309e338. [24] S.Y. Park, T. Yokoyama, N. Shibayama, Y. Shiro, J.R.H. Tame, 1.25 A resolution crystal structures of human haemoglobin in the oxy, deoxy and carbonmonoxy forms, J. Mol. Biol. 360 (2006) 690e701. [25] W.G. Zijlstra, A. Buursma, W.P. Meeuwsen-van der Roest, Absorption spectra
[26]
[27] [28] [29]
[30]
of human fetal and adult oxyhemoglobin, de-oxyhemoglobin, carboxyhemoglobin, and methemoglobin, Clin. Chem. 37 (1991) 1633. D.J. Faber, M.C.G. Aalders, E.G. Mik, B.A. Hooper, M.J.C. van Gemert, T.G. van Leeuwen, Oxygen saturation-dependent absorption and scattering of blood, Phys. Rev. Lett. 93 (2004) 028102. L. Gebicka, E. Banasiak, Flavonoids as reductants of ferryl haemoglobin, Acta Biochim. Pol. 56 (2009) 509e513. N. Ramajunam, K. Vishwanathan, Fluorescence spectroscopy in vivo, in: R.A. Meyers (Ed.), Encyclopedia of Analytical Chemistry, 2011, pp. 20e56. E.C. Liong, Y. Dou, E.E. Scott, J.S. Olson, G.N. Phillips Jr., Waterproofing the heme pocket. Role of proximal amino acid side chains in preventing hemin loss from myoglobin, J. Biochem. 276 (2001) 9093e9100. R. Das, R. Handique, Hemoglobin in Chironomus ramosus (Insecta, Diptera): an electrophoretic study of polymorphism, developmental sequence and interspecific relationship, Hydrobiologia 318 (1996a) 43e50.