Conditions are described under which the catalytic Brdicka currents observed in ammoniacal buffer solutions containing cobalt(III) hexammine chloride or cobalt(II) and very little bovine-serum albumin (0.1-0.2

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Brdic-ka currents observed with bovineserum albumin in ammoniacal buffers with Co(III) and Co(II) groups, like -NH2, -COO-, etc. in their complexation with Co(II). Because of this additional complexity it will remain difficult, if not impossible, to give a quantitative interpretation of the effect of many variables upon the shape and magnitude of Brdicka currents observed with BSA. In our previous paper (1) we presented a tentative mechanism composed of various reactions that take place in the potential range at which Brdicka currents are observed. Continued research has made it evident that this reaction mechanism is incomplete; in this paper we present a modified mechanism that allows at least a qualitative interpretation of the various factors that affect Brdicka currents under conditions of kinetic control. Extensive studies have been made in ammonia, Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol ], and borate buffers on the effects of concentrations of acid and base constituents of the buffers, of pH, ionic strength, calcium and potassium chloride, temperature, and very recently of tetraalkyl salts. Details are being (2) and will be presented elsewhere; in the present paper only a summary of the main results with their qualitative interpretation is reported. EXPERIMENTAL Equipment, chemicals, and performance of experiments have been described (1). Other chemicals not used previously were of C.P. quality. All polarograms were run at 250. Currents were recorded at the moment the mercury drop falls. RESULTSOnly a few results in ammonia, Tris, and borate buffers, which illustrate effects discussed in a later section, are presented in the following figures. In Fig. 1 the ammonium chloride concentration was kept equal to 0.1 MI, but the ammonia concentration was varied. Curves 2a and 3a illustrate the increase of the Brdicka current with increase of pH from 9.2 to 10.3. Curves 2b and 3b illustrate the fact that in the presence of 5 mMi calcium chloride the pattern is changed radically and now two waves are observed. With 0.5 mAM Co(II) instead of Co(III), the polarogram in 1 AM ammonia is very similar to that with Co(III), both in the absence and presence of calcium. This is no longer true in 0.1 AM ammonia, as is evident from a comparison of curves 2a and 2b [Co(III) ] with 4a and 4b [Co(II) l. Fig. 2 illustrates that in a buffer 0.02 MI in ammonium chloride and 0.08 AM in sodium chloride (to keep the ionic strength the same as in Fig. 1) the pattern with Co(III) in 0.1 M ammonia is quite different from that at the same ammonia concentration in 0.1 MI ammonium

In ammonia buffers of varying composition and pH, native bovine serum albumin and completely reduced bovine serum albumin, denoted by G'(SH) 35, yield quite different Brdicka current-voltage (i-E) curves, but they are identical in the presence of 5 mM calcium chloride. This means that in the presence of calcium, bovine serum albumin becomes completely reduced to (P(SH)35. In 8 M urea and ammonia buffers the Brdicka i-E patterns of serum albumin and (P(SH)35 are identical even in the absence of calcium, the effect of calcium on the first wave being negligible while calcium slightly increases the second wave. The maximum polarographic effect is attained at urea concentrations of about 5-6 M. Quite generally, the appearance of a second Brdicka wave is attributed to complexation of Co(II) with S -and a group of ligands that is different on the second than on the first wave. The effect of calcium on Brdicka currents of bovine serum albumin in the absence of urea is attributed to an orientation of the protein on the surface of the electrode such that all disulfide groups are reduced and with' other ligands can complex with Co(II). Denaturation of bovine serum albumin in buffers with a pH less than 10.5, and which are 8 M in urea, is (polarographically) completely reversible if dilution is made within 15 minutes after preparation. Changes in Brdicka i-E patterns upon longer aging at varying pH are attributed-in part at least-to dimerization of the denatured protein by interaction of -S-in one molecule with -S-S-in another.In previous papers (1-4) Brdicka currents observed under various conditions in different buffers with native bovine serum albumin (BSA) and presodium currents (5) with native and modified BSA have been reported and a chain mechanism for the qualitative interpretation of the effects of pH and the kind of buffer on Brdicka currents has been proposed (3). Quite generally it was found that small concentrations of calcium (chloride) as well as magnesium greatly increase the total catalytic currents, or in buffers with two Brdicika waves, the second one, and also affect the shape of current-voltage curves in the voltage range in which Brdicka currents are observed (denoted as Brdicka i-E curves). It appeared of interest to investigate the effect of urea on Brdicka and presodium currents (5). For many years it has been known that urea in concentrations of 6-8 M is a denaturant of proteins, including BSA. Although the interpretation of the mechanism of this denaturation, and particularly the effect of urea on the structure of the protein, is incompletely understood (1-4), which yielded similar polarograms. All chemicals were C.P. products and the same, or of the same quality, as those used previously (1-5).Apparatus and Technique. These were the same as described previously (1-4). Unless stated otherwise, the currents in the presence of larger concentrations of urea have been corrected for viscosity of solutions by multiplying the observed currents by 1.1, 1.2, or 1.3 for 3, 6, or 8 M urea solutio...

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