The interaction of apoconcanavalin A (apo-Con A) with Mn2+ and Ca2+ was studied at 25 degrees C using fluorescence stopped flow. The reaction was monitored using 4-methylumbelliferyl alpha-D-mannopyranoside whose fluorescence is quenched on binding to the metalloproteins. At pH 5.0 entry of Mn2+ into apo is second-order (rate constant = 1.2 x 103 M(-1) s(-1)); at higher pH the rate constant is greater than 104 M(-1) s(-1). Reaction of excess Ca2+ with Mn(Con A) is pseudo-first-order with kobsd = Kk[Ca2+](1 + K[Ca2+])(-1). This is interpreted as rapid formation of unlocked MnCa(Con A), with a formation constant K = 3.5 x 102 M(-1), which transforms (k = 0.026 s(-1)) to a locked form, indistinguishable from native. At pH 6.4 and 7.2, K greater than or equal to 103 M(-1) and k = 0.043 and 0.050 s(-1), respectively. Ca(Con A) and Mn(Con A) precipitate glycogen and bind to 4-methylumbelliferyl alpha-D-mannopyranoside as effectively as native protein at pH 7.2. Treatment of the Ca or Mn forms with EDTA produces an apo form with a small binding capacity, which it loses slowly.

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Communications to the Editor 2849 Prepared in this manner, poly(dimethylphosphazene) bears a superficial resemblance to some lower molecular weight (3500-12 500) products reported by Sisler et al.7 Their compounds were not characterized by NMR and were obtained only in low yields after a tedious purification process from the thermolysis of Me2P(NH2)2Cl. By contrast, our method affords excellent yields and utilizes a conveniently prepared starting material.

The oxidation of deoxy- and oxyhemerythrin to methemerythrin by Fe(CN)6(3-) has been examined. The first step produces (semi-met)O, a half-oxidized form of hemerythrin which has different spectral and kinetic properties from (semi-met)R which is produced by one-electron reduction of methemerythrin. Further oxidation of (semi-met)O to methemerythrin usually occurs only indirectly via disproportionation of (semi-met)O to met and deoxy forms within the octameric framework. Oxidation of (semi-met)R by Fe(CN)6(3-) is a direct second-order reaction. (Semi-met)O is reduced rapidly by dithionite to deoxy, whereas that of (semi-met)R is a slow biphasic process. The oxidation of oxyhemerythrin occurs via the deoxy species, little, if any, reactivity being attributable to the oxy form. The oxidation of the azide adduct of (semi-met)R is biphasic, in which in step one N3(-) is removed during the oxidation to methemerythrin, which in the second step recombines with N3(-). Rate parameters for all these processes at pH 8.2 and 6.3 for protein from Phascolopsis gouldii and Themiste zostericola have been obtained. The implications of these findings to hemerythrin chemistry are discussed.

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