The configuration of NADH and NADPH

It was shown later (in 1955) that some enzymes behave differently from alcohol dehydrogenase these enzymes (e.g., /J-hydroxysteroid dehydrogenase), in fact, utilize 

The configuration of NADH at the 4 position was determined by a chemical transformation to succinic acid [98]. A sample of NADH containing 2H in the A position, 92, was converted to a 6-methoxy derivative, 95 succinic acid was obtained from this derivative by treatment with ozone and then peroxyacetic acid. 

The succinic acid has a (—) ORD curve and was, therefore, (R). In the same way a sample of B -labeled NADH was converted to (+)-(S)-succinic acid. In other words 

Determination of selectivity for a particular enzyme is carried out with the aid of an enzyme of known properties. This is approached by one of two ways  

Although the determination of HA or HB selectivity is relatively straightforward the techniques for isolation of pyridine nucleotides from the reaction mixtures are tedious and time consuming. Two more recent techniques use either proton magnetic resonance or electron impact and field desorption mass spectrometry. The technique of Kaplan and colleagues requires a 220 MHz nuclear magnetic resonance spectrometer interfaced with a Fourier transform system [104], It allows the elimination of extensive purification of the pyridine nucleotide, is able to monitor the precise oxidoreduction site at position 4, can be used with crude extracts, and can be scaled down to /nmole quantities of coenzyme. The method can distinguish between [4-2H]NAD+ (no resonance at 8.95 8) and NAD+ (resonance at 8.95—which is preferred) or between [4A-2H]NADH (resonance at 2.67 8, 75 4B = 3.8 Hz) and [4B-2H]NADH (resonance at 2.77 8, J5 4A = 3.1 Hz). 

---