Lipoprotein-induced phenoloxidase-activity in tarantula hemocyanin

SUPPLEMENTARY MATERIAL Supplementary Figures

Fig. S1. Dopachrome assay of hemocyanin in the presence of hemolymph lipoproteins and dopamine as substrate. Activation of hemocyanin to a phenoloxidase corrected for autooxidation of dopamine. 60 minutes of activation after mathematical correction to OD490 nm = 0 at t = 0 are shown. The increase in enzymatic activity of hemocyanin between 30 and 60 minutes with HDL-2 (blue) incubation is evident. As is the case for activation with HDL-2 + VHDL (red). The effect of VHDL on phenoloxidase-activity is declining from 30 minutes on (green). The data points represent mean values with S.E.M, and second order polynomials are fitted as regression. The protein concentrations were ~300 nM, dopamine was present at a concentration of 10 mM. The absorption was monitored at 490 nm and corrected by division through 0.85 to yield OD490 nm.

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Fig. S2. Dopachrome-assay for the effects of oleic acid on the autooxidation of dopamine. The values shown represent the difference between the autooxidation of dopamine observed in the presence of hemocyanin to the autooxidation observed in the presence of either oleic acid micelles (dark green) or oleic acid monomers (grey).  dopamine turnover = (autooxidation hemocyanin) – (autooxidation oleic acid) Positive values represent a reduced autooxidation in the presence of oleic acid while negative ones indicate an elevated autooxidation in the presence of oleic acid. 60 minutes of activation after mathematical correction to OD490 nm = 0 at t = 0 are shown. The increase in autooxidation suppression from 10 min onward is evident, as is the stronger effect of oleic acid micelles (dark green) on autooxidation of dopamine when compared to the effect of oleic acid monomers (grey). The data points represent mean values with S.E.M, and second order polynomials are fitted as regression. Dopamine was present at a concentration of 10 mM, hemocyanin at ~300 nM, oleic acid at 10 mM (micelles) and 1 mM (monomers). The absorption was monitored at 490 nm and corrected by division through 0.85 to yield OD490 nm. 2

Calculation of the native molecular mass of the lipoproteins

A) Unhydrated density i) Lipids Tab. S1: Unhydrated densities of the different lipid classes present in the Eurypelma lipoproteins. Lipid Cholesterol Cholesteryloleate Trioleylglyceride Oleic acid Phosphatidylcholine

Density / g/mL 1.067 0.997 0.898 0.895 1.032

Reference 1 1 2 3 3

Tab. S2: Density of the Eurypelma lipoprotein lipid moiety. Relative occurrence Lipid

Density / g/mL

HDL-1

HDL-2

Product / g/mL

VHDL

HDL-1

HDL-2

VHDL

Cholesteryloleate

0.997

0.272

0.366

0.129

0.271

0.365

0.129

Cholesterol

1.067

0.024

0.071

0.059

0.026

0.076

0.063

Trioleylglyceride

0.898

-

0.025

-

-

0.022

-

Oleic acid

0.895

0.146

0.082

0.800

0.131

0.073

0.716

Phospholipid

1.032

0.558

0.455

0.012

0.576

0.470

0.012

1.000

0.999

1.000

1.004

1.006

0.920

Sum

ii) Lipoprotein density  Lipoprotein = (Apolipoproteins x relative protein content) + (Lipid x relative lipid content)

(1)

The unhydrated density of the apolipoproteins was assumed to be 1.41 g/mL [4], insertion into (1) yields the unhydrated lipoprotein density : HDL-1:

Lipoprotein = (1.410 g/mL x 0.434) + (1.004 g/mL x 0.566) = 1.180 g/mL

HDL-2:

Lipoprotein = (1.410 g/mL x 0.620) + (1.006 g/mL x 0.380) = 1.256 g/mL

VHDL:

Lipoprotein = (1.410 g/mL x 0.901) + (0.920 g/mL x 0.099) = 1.361 g/mL

Differences to the values published by [5, 6] are to be attributed to the hydration of the lipoproteins in the experimental determination of the density by theses authors.

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B) Partial specific volume  We assumed the partial specific density to be the inverse of the unhydrated density:  = 1/

(2)

Insertion in (2): HDL-1:

 = 1/1.180 g/mL = 0.847 mL/g

HDL-2:

 = 1/1.256 g/mL = 0.796 mL/g

VHDL:

 = 1/1.361 g/mL = 0.735 mL/g

C) Native molecular mass, M According to the Svedberg-equation [7] the molecular mass of a sedimenting particle is defined as: M = (R x S x T)/[D x (1 -  x )]

(3)

With R the universal gas constant (R = 8.3144 JK-1mol-1), S the sedimentation coefficient in 10-13 s, T the absolute temperature in K, D the diffusion coefficient in m2s-1,  the partial specific volume in mL/g and  the density of water at T (H2O, 293 K = 0.998 g/mL).

Tab. S3: Hydrodynamic properties and partial specific volume of HDL-2 and VHDL. Protein HDL-2 VHDL

 / mL/g 0.796 0.735

S / 10-13s 9.7 13.6

D / m2s-1 2.2 x 10-11 2.5 x 10-11

Insertion in (3) yields: HDL-2:

M = (8.3144 JK-1mol-1 x 9.7 x 10-13 s x 293.15 K)/[2.2 x 10-11 m2s-1 x (1 – 0.796 mL/g x 0.998 g/mL)] = 522,899 g/mol = 522,899 Da

VHDL:

M = (8.3144 JK-1mol-1 x 13.6 x 10-13 s x 293.15 K)/[2.5 x 10-11 m2s-1 x (1 – 0.735 mL/g x 0.998 g/mL)] = 496,887 g/mol = 496,887 Da

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D) Theoretical molecular mass of the lipoproteins Mtheoretical Mtheoretical = MAplipoproteins/relative protein content

(4)

Tab. S4: Molecular mass of apolipoproteins, stoichiometry, relative lipid and protein content. Protein HDL-1 HDL-2 VHDL

Molecular mass apolipoproteins / Da 330,000 75,000 250,000 75,000 115,000 95,000

Stoichiometry

Relative lipid content

Relative protein content

1:1

0.566

0.434

1:1

0.380

0.620

2:2

0.099

0.901

Insertion in (4) yields the theoretical molecular mass M theoretical: HDL-1:

Mtheoretical = 405,000/0.444 = 933,180 Da

HDL-2:

Mtheoretical = 325,000/0.620 = 524,194 Da

VHDL:

M theoretical = 420,000/0.901 = 466,149 Da

Literature: [1]

R.C. Weast, CRC Handbook of chemistry and physics., 55th ed., CRC Press 1974.

[2]

S. Schenk, Struktur und Funktion des Lipoproteins von Nereis virens (Annelida), Dr. rer. nat. Thesis, Johannes Gutenberg-Universität Mainz, 2009, p. 193.

[3]

S. Budavari, M.J. O`Neil, A. Smith, P.E. Heckleman (Eds.), The Merck index., 11th ed., Merck & Co., Inc., Rahway, New Jersey, 1989.

[4]

H. Fischer, I. Polikarpov, A.F. Craievich, Average protein density is a molecular-weight-dependent function., Prot. Sci. 13 (2004) 2825-2828.

[5]

N.H. Haunerland, W.S. Bowers, Lipoproteins in the hemolymph of the tarantula, Eurypelma

californicum., Comp. Biochem. Physiol. B 86 (1987) 571-574. [6]

E. Stratakis, G. Fragkiadakis, I. Tentes, Purification and properties of fatty acid-binding VHDL from the hemolymph of the spider Eurypelma californicum., J. Exp. Zool. 267 (1993) 483-492.

[7]

T. Svedberg, N.B. Lewis, The molecular weights of phycoerythrin and of phycocyan., J. Am. Chem. Soc. 50 (1928) 525-536.

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