Acid-catalysed Reaction of Reduced Pyridine Nucleotides

Abstract

IN a recent publication¹ it was demonstrated that the catalysed reaction of DPNH and TPNH in weakly acidic buffers follows first order kinetics, and that the rate of the TPNH reaction at constant acidic pH is considerably greater than that of DPNH. Almost simultaneously, other authors published similar results². The results of a more detailed comparison of the rates for the reduced nucleotides are given in Fig. 1. For TPNH the relationship is log K₁ = 3.71–0.46 pH; for DPNH it is log K₁ = 3.67–0.49 pH, where K₁ is the first-order constant (t = sec.). The figure represents results from experiments using three different lots of enzymatically reduced nucleotides (TPNH, 96.5 per cent DPNH, 99 per cent; Sigma and Co.). For a particular nucleotide no essential difference of rates was noted in the following buffers at equal concentration and pH: phosphate, citrate, tris-hydrochloride, and ethylenediamine tetraacetic acid. It is significant to observe that the two lines converge and, by extension, the rate of the reaction of the two nucleotides in 0.1 N hydrochloric acid theoretically would be indistinguishable experimentally. It is perhaps this which may account for the fact that the difference between the nucleotides has not been observed previously. By extrapolation, the theoretical pH, where the rate becomes zero for both nucleotides, may be obtained. Actually the rate never becomes zero, but the extrapolation may be useful in determining the constants in the overall rate equation. First-order kinetics were obeyed only at low pH. where (H +) is not rate determining, and for small changes in nucleotide concentration.