GEORGEM. FLECK AND ROBERT A. ALBERTY
1678
solutions in presence of sulfuric acid were found to agree within 5% with the values obtained from solubility determinations. Discussion Knowing the mobility a t infinite dilution of the HzS04.HS04- (AHA-) ion to be 65, and that of the proton, SOT3we find a value of AOH+AHA-= 145. It is now possible to obtain K A H A -and the first dissociation constant of sulfuric acid, K13~4, from previously2 determined conductance data of pure sulfuric acid in AN using the plot of A&{1 ~ / K A H Aus. - ]c. This plot yields avalue for KAHA-of 1.03 X lo3 and for K H Aof 5.0 X lo-*, in excellent agreement with the values of 1.15 X lo3 and 4.8 X obtained from spectrophotometrically determined2 equilibrium data of indicators in solutions of sulfuric acid in AN. The value of 1.0 X lo3 for KAHA-obtained in the present paper from solubility data of sodium bisulfate in solutions of sulfuric acid in AN lends strong support to the claim that the assumptions made in the calculations are justified. The stability constant of 1.0 X lo3 for HzS04. HSOI- is considerably greater than those of 1.7 X 102 and 2.8 X 102 for hydrochloric and hydro-
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1'01. 66
bromic acids, respectively. Presumably, strong hydrogen bonding between bisulfate and sulfuric acid occurs in acetonitrile as well as in nitromethane.'
OH
- - -O\sPo01-1/, L o
od
In a discussion of bilateral triple ion formation
3AB 7f ARX+BABin solutions of tetraisoamylammonium nitrate in water-dioxane mixtures, Fuoss8 estimates that XOAB.~X O ~ A B +is equal to one-third to one-half of A ~ A B . This estimate may be valid when dealing with the large size complex ions he used, but it is clear that his estimation should not be generalized for ions of much smaller size. As a matter of fact, the difference between the mobilities of HS04(80) and HzS04.HS04- (65) is remarkably small in acetonitrile.
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(7) H. Van Looy and L. P. Hammett, J . Am. Chem. Soc., 81, 3872 (1959). (8) R. M. Fuoss, ibid., 66, 1857 (1934).
ICIKETICS OF THE REACTION OF PYRIDOXAL AND ALANINE] BY GEORGEM. FLECK^
AND
ROBERT A. ALBERTY
Department of Chemistry, University of Wisconsin,Madison, Wisconsin Recetved M a x h 88, 1968
Kinetics of the reaction of pyridoxal and alanine in aqueous solution have been studied a t 25' by observing the changea in visible and ultraviolet absorbancy and optical rotation which occur following mixing of the reactants. When the initial iilanine concentration is much greater than the initial pyridoxal concentration, the absorbancy, A , due to pyridoxal, inter~ B ~ C - ~where J ~ , ml, m2, and mediates, and products, is given as a function of time by At = BO B ~ e - ~ l B2eyrnnt ma are pseudo first-order constants in the order of decreasing magnitude. Detailed studies are reported of the dependence of one of these pseudo first-order rate constants on pH, initial reactant concentrations, and ionic strength. It is concluded that a t least three compounds which are distinguished spectrophotometrically are formed by the reaction.
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Introduction Pyridoxal reacts with amino acids in a variety of biologically important enzymatic reactions, and analogous non-enzymatic reactions have been reported. It is believed that the aldehyde group of pyridoxal reacts with the a-amino group of the various amino acids to form imines, with subsequent rearrangement of the imine^.^ Several investigators4 have reported absorbancy changes with half-lives of a few minutes when pyridoxal and certain amines are mixed or when the pH of the resulting solutions is changed. The present kinetic study of the reaction of pyridoxal and alanine was conducted to gain in(1) From a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy a t the University of Wisconsin, 1961. This research mas supported by grants from the National Science Foundation, the National Institutes of Health, and the Research Committee of the University of Wisconsin from funds eupplied b y the Wisconsin Alumni Research Foundation. (2) William H. Danforth Graduate Fellow, 1956-1961. (3) E. E. Snell, Vitamins Hormones, 16, 77 (1958). (4) J. B. Neilands and V. Williams, Arch. Biochem. Biophus., 63, 56 (1954); D. E. Metzler. J . Am. Chem. So:., 79, 485 (1957); H. N. Christensen, ibz?. 80, 99 (1958).
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formation regarding the mechanism of the nonenzymatic reaction. Reported in this paper are results of investigations on this reaction, in aqueous solution a t 25.0°, studied by observing changes in visible and ultraviolet absorbancy and optical rotation. Experimental Reagents.-All water used had been redistilled from alkaline permanganate solution in a Barnstead still. The redistilled water was boiled in glass to drive off dissolved gases just before being used to prepare solutions, and precautions were taken to exclude carbon dioxide. For studies of the variation of pseudo first-order rate constant mz with alanine concentration, ionic strength, and pH, C.P. D,L-a-alanine from Pfanstiehl Chemical Co. was used. Alanine from five different lots was separately rrcrystallized from 1 volume of 957, ethanol and 3 volumes of water. The mother liquor was yellow. These recrystallized products were pooled and twice recrystallized from 7 volumes of ethanol and 3 volumes of water. The crystals wcrc washed with ethanol and then dried in a vacuum desiccator over phosphorus pentoxide.6 Alanine for all other spectrophotometric measurements was D, L-a-alanine from Mann Research Laboratories. This product was recrystallized (5) F. J. Gutter and G. Kegeles. abid., 76,3893 (1953); P. I
