Early Stopped-Flow Investigations

Ni2+ was very popular in the early days of the investigation of mechanisms of complex formation, since the time-scale for its reactions with simple ligands was so convenient for the then recently developed stopped-flow technique. However, interest has now moved on to other first-row cations, especially to Cu2+. A review of the kinetics and mechanisms of formation of tetraazamacrocyclic complexes concentrates on Ni2+ and Cu2+, and their reactions with cyclam and similar ligands (267). The tetra(4 -sulfonatophenyl)porphyrin complexes of Ni2+ and of Cu2+ react immeasurably slowly with cyanide, but their IV-methyl derivatives do react, albeit extremely slowly. The relevant time scales are hours for removal of Ni2+, months for the removal of Cu2+, by 10-4 M cyanide at pH 7.4 (268). 

Although three early papers briefly discussed reactions between methylcobalamin and mercury compounds (30-32), the most systematic investigation has come from Wood and co-workers (33). They proposed the mechanism shown in Fig. 1, with values for the various rate constants presented in Table I. Species 2 and 3, in which the benzimidazole nitrogen no longer bonds to the cobalt atom, are termed base-off compounds, whereas 1 is base-on methylcobalamin and 4 is aquocob(III)alamin, the usual product of aqueous transmethylation by 1. Each one of these species has a unique ultraviolet-visible spectrum, which allows quantitative studies by spectrophotometric techniques to be made (28, 32, 33). The mercuric acetate-1 exchange is so rapid that it must be studied using stopped-flow kinetic techniques (33). 

The major disadvantage of all early investigations on chromatographic crossfractionation was related to the fact that both separation modes were connected to each other either off-line or in a stop-flow mode. Regardless of the separation order, SEC vs. HPLC or HPLC vs. SEC, in the first separation step fractions were collected, isolated, and then subjected to the second separation step. This procedure, of course, is very time-consuming and the reliability of the results at least to a certain extent depends on the skills of the operator. 

Following the kinetic and mechanistic study of several steps in the [Fe(NCMe)6] -trimethyl phosphite system some years ago, there has recently been a study of the stepwise replacement of pyridine in [Fe(py)6] by trimethyl phosphite. Although stopped-flow techniques were needed to investigate the early stages, the half-life for conversion of [Fe(py)3(P OMe 3)3] into cis-[Fe(py)2(P OMe 3)4] is a few hours, for further substitution several days. Irradiation of an acetonitrile solution of [Fe(CNMe)4(CN)2] either in the charge-transfer or ligand-field band leads to the successive replacement of two of the four isocyanide ligands by acetonitrile. ... 

The inhibition of carbonic anhydrase by anions has long been recognized. The inhibitory effect of Cl was noted by early investi-gators(19). A later study of anion inhibition by stopped-flow techniques was compromised by the presence of 80mM Cl in buffers used (20). The inhibition of the hydrase activity of the Cobalt(II) substituted enzyme has been investigated over the full pH range(21). Anionic inhibition of esterase activity has been studied by initial rate techniques(11-13) and by complexometric titration(22). None of the work thus far published has included full scale Michaelis-Menten analysis of the inhibition of the native Zinc(II) enzyme towards its natural substrate over an extended pH range. 

---