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RECEIVED for review March 11, 1977. Accepted August 18, 1977. This work was supported by National Science Foundation Grant CHE75-15500.
CORRESPONDENCE Voltammetric Determination of Ultratraces of Albumin, Cysteine, and Cystine at the Hanging Mercury Drop Electrode Sir: During the past few years several authors (1-3) have studied so-called BrdiEka currents observed with albumin and some other proteins ( I ) at the hanging mercury drop electrode (HMDE). These catalytic hydrogen currents were first described by BrdiEka (4)at the dropping mercury electrode and observed with disulfide and/or sulfhydryl-containing proteins (and low molecular weight thiols or disulfides) in ammoniacal buffers containing cobalt(II1)hexammine chloride, [Co(III)], or cobalt(II)chloride, [Co(II)],and only Co(I1) with the low molecular weight compounds. From studies (1-3) at the HMDE it appears that a diffusion controlled irreversible adsorption of albumin (1-3) (and a few other proteins ( I ) ) occurs on the mercury surface from ammoniacal and other buffers. For example, in 3 X M albumin it takes about 2 to 3 min at room temperature to get a monolayer adsorbed; from more dilute solutions, it takes a longer time. The completely or incompletely adsorbed albumin is not desorbed ( I , 3 )upon keeping in ammoniacal buffers, unless a potential more negative than -1.5 V vs. SCE (saturated calomel electrode) is applied. When the rate of adsorption of protein is promoted by stirring, and pure water is used as the solvent, it has been possible to detect and determine as little as lo-’’ M bovine serum albumin (BSA) by measuring the two BrdiEka M currents in 0.1 M ammonia buffer of pH 9.3 which is in Co(II1) or Co(I1). In the present communication, we briefly describe characteristics of a newly discovered catalytic hydrogen current observed at the HMDE on which some “active cobalt” has been deposited and oxidized anodically, followed by cathodic scanning. The procedure allows the detection and estimation of as little as M BSA in 100 mL. The electrodeposition at about -1.0 V (vs. SCE) of “active cobalt” on a HMDE from 0.1 M ammoniacal buffer (pH 9.3) containing a low molecular weight thiol or disulfide and Co(I1) was first described in several papers by Anzenbacher and Kalous ( 5 ) . This “active cobalt” yielded one or two anodic waves with peaks at about -0.25 and -0.05 V. We have observed a large catalytic hydrogen wave with peak at about -1.45 V in 0.1 M ammonia buffer (pH 9.3) containing Co(II1) or Co(I1) and high or low molecular weight thiol or disulfide when the anodic scanning is followed by cathodic scanning. This new catalytic current is denoted in this communication by the symbol i,. The HMDE, the polarographic analyzer and recorder, a magnetic stirrer, the various samples of BSA, as well as purity of chemicals used, have been described previously (3). Unless stated otherwise, all experiments were carried out at 21 “C 2108
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
and with a scanning rate of 500 mV/s. Reported values of the new current, i,, refer to that at the peak potential of about -1.45 V. All potentials refer to the SCE. The buffer used was 0.1 M in ammonia, 0.1 M in ammonium chloride, and 5 X M in cobalt(II1) or Co(I1) (BrdiEka buffer). All solutions used were made and kept air-free during an experiment. The HMDE was placed in the BrdiEka buffer containing a known concentration of BSA, the solution was allowed to stand (or stirred) for a given time and (without stirring) the voltammogram was run from 0 V to -1.05 V, the potential was kept at this potential for 30 s, the scanning reversed to $0.1 V (sometimes to -0.1 or -0.2 V) and after 1 to 2 s the voltammogram run cathodically to -2.0 V. In order to study various factors to be described in a subsequent paper, it was necessary to place the HMDE after adsorption of protein in a protein-free solution in which the scanning was carried out. At a given BSA concentration, adsorption, and therefore i,, increases with increasing time of standing or stirring the solution until about of the mercury surface is covered with protein. Figure 1 illustrates the shape of complete voltammograms obtained in extremely dilute BSA solutions. [For the sake of saving space, the potential range between -0.8 and -0.5 V is omitted in Figure 1.1 After different times of adsorption, the anodic waves exhibited a peak between -0.14 and -0.2 V. At the smallest BSA concentrations, the Co(I1) to Co(0) reduction wave is hardly affected by ultratraces of adsorbed protein; with increasing adsorption, this wave is displaced to less negative potentials (up to 0.2 V) and merges with the wave of the (new) catalytic current, i,. Under conditions of Figure 1the peak potential of i, is between -1.47 and -1.5 V, and when i, is less than 50 g A it is proportional to the concentration of BSA. At larger currents, the plot of i, VI. concentration of BSA is hyperbolic. At larger concentrations of BSA than in Figure 1, a maximum in the hyperbolic curve was found when i, was of the order of 180 FA, to decrease when more BSA was adsorbed on the mercury surface. At concentrations of Co(II1) between 1 and 8 X M, i, has been found proportional to the cobalt concentration; at larger cobalt concentrations, i, became less than proporM Co(III), tional. At concentrations greater than 2.5 X stirring around the mercury surface affected values of i,. The scanning rate has a very large effect on i,. In one set of experiments the following results were obtained: at scan rates of 500, 200,100, and 20 mV/s, peak i, values of 73, 34, 4,
