A Linear Sweep Voltammetric Determination of Proteins With
ThorinWei Sun*, Kui Jiao, Junying Han, Changzhi Zhao
College of Chemistry and Molecular Engineering, Qingdao University of Science
and Technology,
Qingdao 266042 P.R.China,
E-mail: sunwei_1975@public.qd.sd.cn
Absrtract
In pH 3.0 acidic buffer solution,
2-(2-hydroxy-3,6-disulfo-1-naphthyl)-azo-phenylarsenic acid (thorin) can
interact with protein to form a supramolecular complex. The interaction of
thorin with human serum albumin (HSA) was studied in solution by cyclic
voltammetry on mercury electrode. In the presence of HSA, the reductive peak
current of thorin at –0.49 V (vs.SCE) was decreased apparently without the
changes of peak potentials and no new peaks appeared. The electrochemical
parameters of the thorin-HSA interaction solution were calculated and compared
with that of thorin solution, the results showed that there were no obvious
differences in the two sets of parameters. So the formed biosupramolecular
complex was electrochemical inactive and couldn’t take place redox reaction on
the mercury electrode. The binding reaction resulted in the decrease of the free
concentration of thorin in the reaction solution, and the decrease of the
reductive peak current of thorin. The binding constant and the binding ratio of
thorin-HSA were calculated as 1.15×109 and 2:3, respectively. Based on the
decrease of peak current, a sensitive protein assay method was proposed with
second order derivative linear sweep voltammetry. The optimal conditions such as
the effect of pH, the concentration of thorin, reaction time and temperature,
ionic strength et al on the binding reaction had been carefully studied. The
interference of coexisting substances was checked. Under the selected conditions,
the decrease of the reductive peak current of thorin was in proportion to the
concentration of 1.0~12.0 mg L-1 for HSA, 0.1~16.0 mg L-1 for bovine serum
albumin (BSA), 1.0~20.0 mg L-1 for oval albumin (OVA) and 0.4~18.0 mg L-1 for
lipase et al. This new electrochemical method was further applied to the
determination of human serum samples and the results were in good agreement with
the traditional Commassie Brilliant Blue G-250 (CBB G-250) spectrophotometric
method.
Key words: protein, thorin, linear sweep voltammetry, interaction