Interaction between pyridine nucleotide coenzymes and heme proteins as a possible source of error in assay of activities of coenzyme-linked enzyme.
The ultraviolet absorbance spectra of pyridine nucleotide coenzymes change in the presence of heme-containing proteins. The positions of each of the two main absorbance peaks of NADH are shifted progressively towards shorter wavelengths in the presence of increasing concentrations of hemoglobin, and the third peak, at 220 nm, disappears altogether. Similar changes are seen in the spectra of NAD+ and NADPH, and similar effects on these spectra are produced by myoglobin and cytochrome c, but not by comparable concentrations of albumin. The spectral shifts are generally accompanied by a decreased peak height. This finding may help explain problems reported by previous workers in the measurement of the activity of enzymes such as transketolase or lactate dehydrogenase in erythrocyte hemolysates. Errors may be considerable if allowance is not made for this effect, especially if the concentration of heme protein in the spectrophotometer cuvette much exceeds 1 g/L. The interaction appears to indicate some form of bonding, occurring generally between pyridine nucleotide coenzymes and the heme group in proteins. We relate the findings to measurement of activities of pyridine nucleotide-linked enzymes in erythrocyte lysates and in plasma containing myoglobin after muscle breakdown.
Jeyasingham
M D
MD
Department of Neuropathology, Institute of Psychiatry, London, U.K.
Pratt
O E
OE
Roopral
H K
HK
D003067:Coenzymes / Q000032:analysis*; D003574:Cytochrome c Group / Q000032:analysis; D003951:Diagnostic Errors; D004912:Erythrocytes / Q000032:analysis; D006420:Hemeproteins / Q000494:pharmacology*; D006454:Hemoglobins / Q000494:pharmacology; D006801:Humans; D009211:Myoglobin / Q000494:pharmacology; D009243:NAD / Q000032:analysis*; D009249:NADP / Q000032:analysis*; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D003067:Coenzymes; D003574:Cytochrome c Group; D006420:Hemeproteins; D006454:Hemoglobins; D009211:Myoglobin; D009243:NAD; D009249:NADP