Hydride Ion and Sulfite Ion Attack on Pyridine Derivatives Correspondence between Biochemical and Chemical Reactions as Demonstrated by UV-Spectroscopy Peder Olesen Larsen. Maria Sundahl. and Elibieta Wleczorkowska Royal Veterinary and Agricultural University DK-1871 Copenhagen, Denmark T h e nicotinamide coenzymes, NAD+/NADH and NADP+/NADPH, are intermediates in the majority of biochemical oxidation-reduction processes. Formally, the reduction of the oxidized forms of the coenzymes involves transfer of a hydride ion from the substrate, for example from a n alcohol, although a free hydride ion is not present at any moment. H
+
H
R
The reduced forms of the coenzymes, NADH and NADPH, have a specific absorption band at 340 nm, not present in the oxidized forms. Measurements of the absorption a t 340 nm is used for a multitude of enzyme assays and in quantitative enzvmatic determinations of manv substrates. All students of l&hemistry need therefore to he familiar with the nicotinamide coenzvmcs and with the chemistrv involved in their
1080
Journal of Chemical Education
The following experiments have been developed for our undergraduate biurhemistry course with this p u 6 m e in mind and, furthermore, to familiarize the students with UV-spectroscopy as a tool for investigation of reaction mechanisms in organic chemistry and biochemistry. The specific questions asked are 1) Can the hydride ion transfer only be performed in enzymatic reactions or also in non-enzymaticreactions with proper hydride ion donors? 2) Can nucleophiles other than hydride ions add to the NAD+system? 3) What part of the NADt-molecule is necessary to give the chemical as distinct from biochemical reactivity? 4) What part of the NAD+-moleculeis necessary to give reactivity in an enzyme-catalyzed reaction (how specific is the enzyme for NAD+)?
Answers to these questions are found by UV-spectroscopy using NAD+ (I), nicotinic acid (II), N-methylnicotinic acid amfoion (trigonelline) (III), nicotinamide (IV), and 1methylnicotinamide ion (N'-methylnicotinamide ion, 3-carbamido-l-methylpyridiniumion) (V) as substrates and ethanol alcohol dehydrogenase (E.C. 1.1.1.1) or sodiumborohydride or sulfite ions as reagent.
+
are listed in the Table. The students may very well experience difficulties in obtaining correct €-values.These difficulties are mainly
due to weighing errors (unless large quantities of the rather expensive NADf are used) and dilution errors. On the other hand, A,, values can easily he precisely determined. Discussion The results show that reduction of NAD+ can be performed both enzymatically and non-enzymatically. With sulfite, a similar process is raking place as judged from the appearance of an absorption peak of 320 nm. Sulfite is known as a nucleophile in the addition to carhonyl compounds.
I n a single experimental period of 3 h r not all the combinations of suhstrates and rewents can be used. However, the individual students and gro;ps of students can use different suhstrates, and in this way rhecomhined experiments of the whole class can give a complete set of data. Experimental Procedure Alcohol dehydrogenase, NADC,nicotinic acid, nicotinamide (niacinamide), N-methylnicotinic acid hydrochloride (trigonelline hydrochloride),and l-methylnicotinamideiodide can he obtained from various biochemical suppliers, including Sigma Chemical Compan) A recording spectrophutometer, Perkin-ElmerModel 402, wm uspd with l.cm silica cells in our lahoratory, but any recordmg sperm,phoromcter wdl du. Meaxrernenrs were made betwepn 231)and390 nm. UV-data for the substrates can be found in the literature (compare Table). It provides good exercise for the students to find the data for NADt and NADH themselves. For the other suhstrates the preliminary assumption can he made that their spectrwcopic properties are similar to those of NADt (although this assumption neglects the contribution from adenine in the NADf; therefore, in fact, the extinction coefficientsat 260 nm differ by a factor of 5). Prepared in advance are 0.1M tris-acetatehuffer pH 8 and sodium sulfite solution, 1 M adjusted to pH 8. A stock solution of NAD+ in the huffer is prepared so that it, by dilution 1:30, gives absorbance between 0.5and 0.7 at 259 nm. Then 100p1 of the stack solution is added to 2.9 ml of buffer in the UV-cell and the UV-spectrum recorded against huffer. Next, 500 pl of the stock solution is added to 2.5 ml of huffer and the spectrum again recorded to show the absence of absorbance ahove 3Ml nm.Afterwards 20 ma solid NaBH4is added and after shaking the spectrum ismeasuredagain after evolution of gas has ceased. Next, 500 p1 of the NADC-stacksolution is added to 2.5 m10.5 M ethanol in tris huffer. nH 8. After the spectrum is measured, 100 r l of solution containing 25 pg alcohol dehidrogenase from yeast in the huffer is added, and the spectnun is recorded again 5 and 15 min after
.. - - ...-.....-. -...., ...- ...
T h e lack of reaction with nicotinic acid and nicotinamide shows that the quarternized positively charged nitrogen atom in the pyridine ring is necessary for nucleophilic attack on the ring. The group substituted on this N is important. NAD+ can react with both hydride ions and sulfite, whereas the N-methyl derivative 111 only reacts with hydride ion under the conditions described here. N-methylnicotinic acid is not attacked ~ conditions hy any of nucleophiles under t h experimental chwen here. This demonstrates the importance of an electron withdrawine suhstituent in the ." ~ v r i d i n erine. " A1 ~. 1 81 the carboxyl group will be present as the carboxylate ion. T h e enzymatic reaction can use only NAD+ as substrate. This shows the importance of the substituent on the N-atom in the ~ v r i d i n rei m in determininn enzvmatic specificitv. he-Cterature contains many repor& on t h e addition of nucleophiles to NAD+ and related substances, for example on the addition of sulfite ( 2 ) and of cyanide (4,6). NAD+ is more prone to nucleophilic attack than l-methylnieotinamide It has been shown that N-methylnicotinicacid can he ion (i). reduced with sodium dithionite underappn~priateconditions and its lack of reactivity toward nucleophiles is thrrefore not complete (4). Likewise, it has been shown that cyanide can react with this compound in almholic solution, although it will not react in water to any considerable degree ( 4 ) . Cyanide reacts easilv with NAD7 and N.methvlnicotinamide ion in water (4,6f, but sulfite has been chosen in the experiments here to avoid the use of ~oisonouscvanide solutions. and because a cyanide experiment could nbt be performed in a buffered solution at DH 8. where all cvanide would br Present as hydrocyanic acid
-
",
Finally. SMlpl ofthe YAD--solution isadded to 2 ml of bufferand 500 rrl ufthe anlium sulfite solutiun.'Che spwrrum is recorded against 5 0 6 4 sulfite solution and 2.5 ml huffer. The same series of experiments is repeated with the other suhand r,. values and strates. The data are used for tabulationif A, for conclusions about enzymaticreduction, chemical reduction with NaBH4, and sulfite addition. UV-data from the literature and conclusions from the experiments
Literature Cited (1) D a m n . R. A. C.. Elliott, D. C., Elliott, W. H., and Jon-, K. M. (Editors1 Data for Biahemieal Research. 2nd Ed.. 1969. Sann,E., and Stoek, A,, Chem. Bar.93,3083 (1960). ( 3 ) Hughes, E. B., Jellinek. H. H. G.. and Ambmse. B.A,. J, Phys. Cham.. 53.414 (1949). (4) Lambore. M. R,Burton, R. M., and Kepian, N. 0.. J. Amer. Cham. Soe. 79,6173 (2) Pneiderer, G.,
(1957). (5) Jellinek, H. H. G. and Wayne, M.G.. J Phys. Chsm.. 55,173 (1951). (6) Colowiek, S. P., Ksplan, N. 0.. and Ciotti, M. M., J. B i d Chem., 191,447 (1951)
UV-Data for and Reactlvlty cd MAD+ and Related Compmnds Oxidized form Amx
(nm) NAD+
11)
Nicotinic acid (11) N-Methylnicotinic acid hydrochloride (Ill) Nicotinamide (IV) 1-Melhylnicotinamide
Reduced form
ern
(nm)
(n
17800 ( l~
261.5 (3) 265 (4)
2960 (3) 9300 (4)
355 (4)
261.5 (5) 265 (4)
2890 (5) 4100 (4)
365 (4)
259
Reduction
with Alcohol
rn
259(lJ 338 113
ern
dehydrwenase
ISSOO(0
6220 ( 0
Volume 59
NaBHl
Addition of sulfite A(nm)
+ Number 12 December 1982
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