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TitleAqueous Two-Phase Systems - R. Hatti-Kaul (Humana, 2000) WW
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Page 1

M E T H O D S I N B I O T E C H N O L O G Y � 11TM

HUMANA PRESS

Aqueous
Two-Phase

Systems

Edited by

Rajni Hatti-Kaul

Methods and Protocols

Aqueous
Two-Phase

Systems
Methods and Protocols

Edited by

Rajni Hatti-Kaul

Page 2

Aqueous Two-Phase Systems

Page 227

212 Franco

3.2. Partitioning of Proteins in ATPS

1. Stock solutions of PEG, Dextran or other materials (phase forming salts) are
weighed into flasks with solid NaCl being added when necessary to give the
desired composition (described in Tables 1–3).

2. Fine adjustments of pH are made if required with NaOH or HCl to give the desired
values (see Note 12).

3. Protein solutions are mixed with the ATPS using a Vortex mixer for 1 min. Pro-
tein concentration in the systems should be approx 1 mg/mL of total volume. At
least three replicates are done for each sample.

4. The volumes are then made up (5.0 mL) with distilled water as required.
5. Phase separation is achieved by centrifugation for 3 min at 3000g.
6. Phases are carefully separated and the interface of each tube is discarded.
7. Determine the concentration of the proteins in the phases (see Note 13).
8. Partition coefficients (K) of the modified proteins are found by calculating the

ratio of protein concentration in the top phase to that in the bottom phase.
9. Resolution of the ATPS for protein surface hydrophobicity (Rh) is defined as the

slope of the lines obtained by plotting the values of log K (log of the partition coeffi-
cient of the hydrophobically modified proteins) vs log P (the unit of hydrophobicity
used: 2 M minus the value of (NH4)2SO4, or [2 M – Me]) (see Notes 4 and 14; Fig. 1).

10. Resolution of the ATPS for protein surface charge (Rc) is defined as the slope of
the lines obtained by plotting the values of log K vs the charge density of the
charged modified proteins (see Note 15 and Fig. 2).

4. Notes
1. For hydrophobic modification, it is preferred to choose proteins with low surface

hydrophobicity, as measured by HIC, because the method of chemical modifica-
tion used increases protein hydrophobicity and it is desirable for all the modified
products to elute from the HIC column in a suitable decreasing concentration
linear salt gradient. The proteins for this study, BSA and -lactoglobulin, have
also the additional advantage of having average (i.e., not extreme) isoelectric
points (4.8 and 5.1, respectively).

2. In order to have proteins with different hydrophobicities, four different groups
were inserted: one with 6 carbons (hexanoic), one with 4 carbons (butyric), and
one also with 4 carbons and a free carboxylic group (succinic), and acetic anhy-
dride with only 2 carbons. According to reactions 1 and 2 (below) some of the
free amino groups of the protein polypeptide chains are substituted by an ali-
phatic chain donated by the anhydride.

Protein + Monocarboxylic anhydride (e.g., hexanoic)

P-NH2 + (CH3(CH2)4CO)2O PNH-CO(CH2)4CH3 + CH3(CH2)4COOH (1)

Protein + Dicarboxylic acid anhydride (internal anhydride, e.g., succinic)

PNH2 + (RH-COO-CO)2O PNHCO-R-COOH (2)

This method can be used for most proteins by considering specific requirements
in each case.

Page 228

Partitioning of Chemically Modified Proteins 213

Table 2
Composition of ATPS Used for Partitioning
of Hydrophobically Modified BSA (Systems pH 5.3) (4)

Phase component 1, % Phase component 2, % NaCl, % Ra

PEG 1500 20.0 Phosphate, 14.0 — 20.7
20.0 Phosphate, 14.0 6.0 44.0

PEG 4000 11.0 Phosphate, 15.0 — 12.0
11.0 Phosphate, 15.0 3.0 11.0
11.0 Phosphate, 15.0 6.0 20.4

aValues of resolution of ATPS for differences in protein surface hydrophobicity.

Table 1
Composition of ATPS Used for Partitioning
of Hydrophobically Modified -Lactoglobulin (Systems pH 5.3) (4)

Phase component 1, % Phase component 2, % NaCl, % Ra

PEG 1500 15.0 Phosphate, 10.0 — 10.2
15.0 Phosphate, 10.0 6.0 25.5
15.0 (NH4)2SO4, 15.0 — 9.1
15.0 MgSO4, 15.0 — 3.5
15.0 Na citrate, 15 — 11.4

PEG 4000 15.5 Phosphate, 15.2 — 22.6
14.7 Phosphate, 14.7 6.0 29.0
7.0 Dextran, 7.0 — 6.0
7.0 Dextran, 7.0 3.0 5.0
7.0 Dextran, 7.0 9.0 8.0

aValues of resolution of ATPS for differences in protein surface hydrophobicity.

Table 3
Salt Composition of the ATPS Systems in 7% PEG 4000/
7% Dextran T500, pH 7.9, for Measuring the Resolution
for Differences in Protein Surface Charge (5)

Buffer Salt

50 mM phosphate 50 mM Li2SO4
50 mM phosphate None
50 mM phosphate 50 mM Na2SO4
50 mM phosphate 200 mM NaCl
50 mM phosphate 50 mM MgSO4
50 mM Tris None
50 mM phosphate 500 mM NaCl
50 mM phosphate 100 mM Na2SO4
50 mM phosphate 100 mM KCl

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