Supplementary Data s9

1Supplementary Data

3Functional characterisation of the haemoglobins of the migratory notothenioid fish

4Dissostichus eleginoides

6Daniela Coppola1, Daniela Giordano1, Stefania Abbruzzetti2, Francesco Marchesani3, Marco

7Balestrieri1, Guido di Prisco1, Cristiano Viappiani4, Stefano Bruno1,3*, Cinzia Verde 1,5*

91 Institute of Biosciences and BioResources, CNR, Via Pietro Castellino 111, I-80131 Naples, Italy

102 Dipartimento di Bioscienze, Università degli Studi di Parma, Parco Area delle Scienze 11A, 43124

11Parma, Italy and NEST Istituto Nanoscienze, CNR, Piazza San Silvestro 12 - 56127 Pisa, Italy.

123 Dipartimento di Farmacia, Università di Parma, Parco Area delle Scienze 23/A, 43100 Parma, Ital

144 Dipartimento di Fisica e Scienze della Terra, Università degli Studi di Parma, Parco Area delle

15Scienze 7A, 43124 Parma, Italy and NEST Istituto Nanoscienze, CNR, Piazza San Silvestro 12 -

1656127 Pisa, Italy.

175 University “Roma 3”, Department of Biology, Viale Marconi 446, I-00146 Rome, Italy.

19*Corresponding authors:

20Cinzia Verde

21E-mail: [email protected]; [email protected]

22Stefano Bruno

23E-mail: [email protected]

27Figure S1. Ion-exchange chromatography of D. eleginoides hemolysate. A Mono Q-Tricorn 28column (1.0 x 10 cm) was equilibrated with 20 mM Tris-HCl pH 8.0 (Buffer A). Elution was 29performed with a gradient from Buffer A to Buffer B (500 mM NaCl in Buffer A) (dash line). Hb1 30and Hb2 were eluted at 20 and 25% Buffer B, respectively. 31 32 33 34 35 36Figure S2. Oxygen-equilibrium isotherms measured in T. bernacchii Hb1 in 100 mM Hepes, pH 377.5 (closed circles), and 100 mM Hepes, 100 mM NaCl, 3 mM ATP, pH 7.5 (closed triangles). All 38experiments were performed at 5°C. 39

43 44Figure S3. CO-rebinding kinetics to D. eleginoides Hb1 (A) and Hb2 (B) solutions equilibrated 45with 1-atm CO (grey) and 0.1-atm CO (grey) in the presence of 100 mM NaCl, 3 mM ATP. Black 46curves are the result of a fit with a sum of two stretched exponential and two exponential 47relaxations. 10°C, pH 7.5. 48 49 50 51 52 53 54 55Table 1S. Sequence identity (%) of  (A) and (B) chains of Hbs of notothenioids, Arctic and temperate fish. The  and  chains of D. eleginoides Hbs are in grey. The abbreviations on top of the columns (Ca, Ee, etc) from left to right, correspond to the species from bottom to top. Adapted from Verde et al., 2008. The list of names of species are in Table 1, with the exception of Gadus morhua and Boreogadus saida. 57 A.  chains

Identity (%) C E H S A A C T N R Amy Species Ccl a e l s a a c Om Am Am Gm De n Gg c Dm g Ps Ga Bm Ao t Pu Cm Gg Na Nc Bd Bd Em Tn Hemoglobin /Globin C A C IV 1 2,3 2 1,2 2 2 2 1,2 1,2 1 1 1 a2 a1 1,C 1,C

T. bernacchii Hb1,C 59 64 59 56 54 54 52 63 61 66 74 59 90 62 64 66 92 92 88 90 90 87 90 91 91 94 96 89 80 66 88 97 T. newnesi Hb1,C 58 62 57 55 63 53 52 61 61 64 72 60 89 63 64 66 90 90 85 91 88 85 90 76 90 92 93 87 78 64 86

E. maclovinus Hb1,C 61 66 61 59 55 57 54 67 60 68 74 62 85 86 67 69 85 85 84 83 85 84 85 82 84 88 88 81 84 70 B. diacanthus α1 60 66 62 63 62 57 63 68 62 82 68 65 65 82 84 86 65 68 66 64 68 66 65 66 66 66 65 62 69

B. diacanthus α2 60 64 64 58 60 53 56 64 57 71 77 61 79 65 66 67 80 77 76 75 79 76 76 79 76 79 78 73 N. coriiceps Hb1 55 61 54 53 51 50 51 59 57 62 68 56 83 61 61 62 85 86 83 81 83 83 83 71 83 86 90

N. angustata Hb1 59 66 60 56 57 54 53 64 61 67 75 59 90 64 65 66 91 93 90 88 92 90 91 77 89 95 G. gibberifrons Hb1 59 63 59 56 55 53 52 63 61 69 75 61 87 65 67 68 88 89 87 89 91 86 87 78 90

C. mawsoni Hb1,2 58 63 57 56 54 53 52 63 61 64 72 61 84 64 66 69 85 88 85 92 88 84 84 77 P. urvillii Hb1,2 57 63 62 61 58 54 57 61 58 66 76 57 76 63 64 65 77 76 75 75 76 74 72

A. mitopteryx 58 61 56 53 53 53 50 61 58 64 70 61 89 64 61 63 89 88 84 85 85 84 A. orianae 61 66 62 58 58 56 53 66 59 66 73 58 85 64 65 66 86 90 96 81 89

B. marri 59 65 61 57 59 54 55 64 63 68 74 62 84 68 70 71 85 94 90 86 G. acuticeps 57 61 55 54 54 52 52 61 61 64 71 59 84 65 64 66 85 86 82

P. scotti 61 66 63 58 58 54 54 66 59 68 73 58 85 64 66 66 87 90 R. glacialis 59 67 60 57 59 57 54 65 61 67 73 60 89 65 66 67 89

D. mawsoni α 62 63 59 56 56 53 54 61 60 65 72 58 96 62 66 64 N. coriiceps Hb2 56 65 61 60 61 56 61 65 63 80 68 66 63 93 95

G. gibberifrons Hb2 56 65 61 61 62 57 61 65 61 81 68 66 64 95 T. newnesi Hb2 55 64 59 61 63 57 61 64 61 57 66 66 60

D. eleginoides Hb1,2 62 61 61 56 56 51 53 59 59 66 72 57 G. morhua Hb2 59 64 61 56 58 52 55 64 62 65 58 A. minor Hb2,3 61 69 64 63 62 57 56 67 59 72 A. minor Hb1 59 66 64 67 69 57 64 66 61 O. mykiss HbIV 63 68 65 58 63 60 54 66 C. carpio 81 90 73 64 66 67 62 A. anguilla HbC 54 64 59 68 69 54 A. anguilla HbA 61 66 61 56 57 S. salar 62 67 69 73 H. littorale HbC 58 63 63 E. electricus 66 76 C. auratus 76 C. clarkii 58 59 1 5 60

Bchains Identity (%) Amy Species Ca Ee Hl Aa Aa Cc Om Am Am Bs Gm De De Tb Tn Pu Gg Cm Dm Rg Ps Ga Bm Ao t Pu Cm Gg Na Nc Bd Bd Em Em Tn Hemoglobin C A C IV 3 1,2 1,2 2,3 2 1 C C 2 2 2 1 1 1 1,2 1,2 β2 β1 C 1 1,2 T. bernacchii Hb1 63 59 56 55 60 60 65 76 75 60 71 70 90 69 69 69 68 66 90 84 78 82 86 80 85 81 88 95 91 88 73 82 71 81 93 T. newnwsi Hb1,2 59 56 54 55 57 56 60 72 75 57 70 69 84 67 67 67 66 64 87 82 77 80 84 78 82 77 84 89 84 84 71 80 69 76 E. maclovinus Hb1 65 63 54 56 64 63 64 75 81 63 71 73 80 72 71 71 71 67 79 77 73 76 77 76 78 82 78 81 82 80 72 83 70 E. maclovinus HbC 62 53 53 57 58 59 63 80 64 64 71 87 69 90 89 86 86 82 71 66 64 65 67 67 67 67 67 72 71 71 84 67 B. diacanthus β1 62 60 53 57 64 61 63 71 75 61 70 71 82 70 69 69 70 67 82 76 75 72 77 76 74 83 77 80 80 79 72 B. diacanthus β2 63 57 54 57 63 60 67 86 69 66 71 86 74 86 86 90 86 83 75 67 69 68 69 71 70 71 69 74 73 72 N. coriiceps Hb1,2 58 56 56 54 60 57 63 75 78 58 69 70 88 70 70 69 68 65 85 86 82 80 89 82 81 78 87 89 93 N. angustata Hb1,2 60 57 58 54 60 58 63 76 78 60 71 76 91 70 70 70 69 65 89 87 82 80 89 83 84 80 87 93 G. gibberifrons Hb1 62 61 57 55 60 59 65 77 77 60 71 71 91 71 71 71 69 67 90 85 80 82 86 81 84 78 88 C. mawsoni Hb1 58 56 53 54 59 55 61 73 75 58 70 68 86 68 68 67 66 67 83 89 83 84 87 82 80 76 P. urvillii Hb1 64 59 54 56 60 62 65 71 77 64 71 69 81 69 68 68 67 65 81 76 72 74 76 73 76 A. mitopteryx 57 55 56 54 59 56 59 71 78 58 68 68 84 66 67 66 67 62 85 79 75 78 79 76 A. orianae 58 56 55 53 58 57 61 71 74 60 69 68 82 67 67 67 68 63 81 80 90 77 81 B. marri 58 54 55 54 59 56 60 73 76 59 67 67 85 67 67 67 67 65 83 87 78 84 G. acuticeps 58 56 53 54 58 56 58 70 71 61 65 66 79 66 67 65 65 65 79 79 78 P. scotti 57 57 54 52 57 56 60 69 73 60 67 65 82 65 65 65 65 62 81 80 R. glacialis 56 54 54 53 56 54 60 71 75 57 68 65 85 66 65 66 65 61 83 D. mawsoni β 63 61 57 55 61 59 65 76 77 60 72 70 95 70 70 71 70 66 C. mawsoni Hb2 57 54 48 56 60 54 59 78 71 58 67 89 65 89 89 84 86 G. gibberifrons Hb2 63 54 53 59 58 60 63 82 68 65 73 92 69 91 89 87 P. urvillii Hb2 60 53 51 56 60 57 63 84 68 64 71 87 71 88 86 T.newnesi HbC 60 54 52 56 60 58 61 81 66 61 68 93 69 95 T. bernacchii HbC 61 54 52 56 58 58 63 82 75 62 70 94 69 D. eleginoides Hb1 61 59 57 57 60 58 65 78 78 60 58 70 D. eleginoides Hb2 61 54 53 57 62 58 63 81 66 63 68 G.morhua Hb2,3 63 56 52 58 58 60 63 72 71 62 B.saida Hb1,2 64 56 58 59 61 64 61 65 60 A. minor Hb1,2 63 58 57 56 59 60 63 73 A. minor Hb3 63 56 54 57 58 61 64 O. mykiss HbIV 78 70 58 72 60 72 C.carpio 91 68 60 66 59 A.anguilla HbC 64 63 69 56 A.anguilla HbA 71 59 50 H.littorale HbC 62 59 E.electricus 72 C.auratus 61

2 6 62 63Table S2. Results of the individual fittings of the CO-rebinding kinetics to D. eleginoides Hb1 and Hb2 with the equation    t  1  t  2  t   t          64  1    2    3    4  A  A1e  A2e  A3e  A4e 65in the absence and presence of allosteric effectors (100 mM NaCl, 3 mM ATP) at 1-atm CO and 0.1-atm CO. 10°C, pH 7.5. 66

[CO] A1 1(ns) 1 A2 2 (s) 2 R (%)  3 (ms) T (%)  4 (ms) (atm) Hb1 1 0.30±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 281 0.74±0.03 722 6.8±0.1

+ATP, 0.28±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 161 0.79±0.05 842 16.5±0.2 Cl- 0.1 0.30±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 151 5±1 852 50±5

+ATP, 0.28±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 71 6.4±0.8 932 100±10 Cl- Hb2 1 0.33±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 221 0.5±0.1 782 5.8±0.1

+ATP, 0.30±0.01 40±10 0.34±0.05 -0.04±0.01 20±5 0.5±0.05 161 0.5±0.1 842) 12.7±0.1 Cl- 0.1 0.33±0.01 40±10 0.34±0.05 -0.05±0.01 20±5 0.5±0.05 111 5±1 892 75±1

+ATP, 0.30±0.01 40±10 0.34±0.05 -0.04±0.01 20±5 0.5±0.05 81 3.5±0.8 922 72±1 Cl- 67 68