Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Kurek et al. (2002) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.162077099

Table 1. Analysis of zinc binding to glutathione S-transferase
(GST) and GST-A1N₁₇₆

Sample	Zn ppb (nmol/10 ml)	Protein-bound Zn, mol Zn/mol protein
Buffer	2.2 (0.33)	–
GST, 1.36 nmol	15.3 (2.32)	1.46
GST-A1N₁₇₆, 1.55 nmol	45.6 (6.91)	4.25 (2.06 + 2.19)*

To quantify Zn²⁺ binding, purified GST (1.36 nmol) and GST-A1N₁₇₆ (1.55 nmol) were dialyzed against 1 mM Hepes–KOH, pH 7.6, and then diluted with the dialysis buffer to 10 ml. Zn²⁺ concentrations were measured by using an inductively coupled plasma-mass spectrometer [PE Sciex (Thornhill, Ontario, Canada) ELAN 6000] with Rh³⁺ ion as an internal control at the laboratories of the Geological Survey in Jerusalem, Israel. ppb, parts per billion.

*Specific Zn²⁺ content in GhCesA1-N was calculated by subtracting estimated nonspecific binding assuming that GST binds Zn ions nonspecifically and that the average number of Zn ions bound nonspecifically on the surface of the proteins is proportional to the surface area. Thus, for GST-A1N₁₇₆, 4.25 mol/mol protein includes Zn²⁺ bound on the protein surface. Because GST-A1N₁₇₆ (41 kDa) has a larger surface area than GST (29 kDa), of the 4.25 mol Zn²⁺ bound, 2.06 mol is considered nonspecific surface binding, leaving an estimated 2.19 mol of Zn²⁺ ions bound specifically by the Zn-binding domains.