TABLE 1.

CzcD retention by metal affinity chromatographya

Metal cationPretreatment of CzcD% Retention
Zn2+None100
Co2+None100
Cu2+None100
Ni2+None87
Cd2+None0
Mn2+None0
Mg2+None0
Zn2+Zincb75
Zn2+DEPCc0
Zn2+DEPC/hydroxylamined75
  • a Chelating fast-flow Sepharose columns were loaded with several heavy metal cations and used for metal affinity chromatography with purified CzcD protein. After the protein was applied and washed with several bed volumes of 10 mM Na phosphate buffer (pH 7.2) containing 0.5 M NaCl, CzcD was eluted with the same buffer containing 10 mM EDTA. The fractions were collected and analyzed by polyacrylamide gel electrophoresis (original data are shown for Zn2+ and Mg2+ in Fig. 4). The gels were scanned, and the proportions of total CzcD in the wash and elution fractions were determined and used to calculate the percent retention.

  • b The CzcD protein was incubated in the presence of 1 mM Zn2+ for 30 min at 0°C prior to chromatography.

  • c The CzcD protein was incubated in the presence of 1 mM Zn2+ for 60 min at 0°C with DEPC at a concentration that was two times the concentration of histidine residues of CzcD.

  • d The CzcD protein was incubated in the presence of 1 mM Zn2+ for 60 min at 0°C with DEPC and thin for 12 h at 0°C in the presence of 75 mM hydroxylamine.