Absorption spectrum, coenzyme

In the present experiment, you will determine the activity of isocitrate dehydrogenase extracted from pork heart muscle. The commercial preparation comes in powder form and it uses NADP+ rather than NAD+ as a coenzyme. The basis of the measurement of the enzyme activity is the absorption spectrum of NADPH. This reduced coenzyme has an absorption maximum at 340 nm. Therefore, an increase in the absorbance at 340 nm indicates an increase in NADPH concentration, hence the progress of the reaction. We define the unit of isocitrate dehydrogenase activity as one that causes an increase of 0.01 absorbance per min. at 340 nm. 

The optical changes that occur after complex formation between the enzyme and the coenzyme are expressed in the fluorescence spectrum. The excitation spectrum is usually reflected in the absorption spectrum, since the energy taken up in the absorption region is generally emitted at another wavelength. 

The cobalamin coenzyme is bound by the apoenzyme with no significant change in the absorption spectrum." This suggests that no major change occurs in the coordination of cobalt(IIl). The first step of the reaction involves homo-lytic fission of the Co—C bond 2-184, 186-188... 

The equilibrium and rate constants for NADH binding to the three isozymes EE, ES, and SS of the horse enzyme have been determined 305). Differences in binding to the two types of chains were found both for the binding strength and the pH dependence. Changes in the absorption spectrum 306,307), the fluorescence polarization spectrum (305), the optical rotatory dispersion spectrum 309), and the effect of DzO on the fluorescence spectrum 310) have been studied for the binary enzyme coenzyme complexes compared to the free molecules. 

Subsequently, Cross et al. (SOI) demonstrated that formation of the E-NADPH binary complex, and the abortive ternary complexes E-NADPH-L-glutamate and E-NADP-a-ketoglutarate are all characterized by a red shift in the tryptophan absorption spectrum. It appears likely, therefore, that a tryptophan residue is located in or near the coenzyme binding site. 

An alternate and competing pathway for the metabolism of cysteinesulfinic acid is its oxidation to cysteic acid (reaction 7, Fig. 2). A new enzyme, L-cysteinesulfinate dehydrogenase, has been isolated from P. vulgaris which catalyzes this reaction. Its unique property is that it is reported to require a hitherto unknown coenzyme, designated as Co III, for activity. A good source of Co III is boiled yeast extract. The reduced form of the coenzyme has an absorption spectrum characteristic of dehydropyridine nucleotides. The structure of this supposed new coenzyme is uncertain. The fact that it can substitute for DPN in many reactions supposedly specific for the latter leads to some wonderment. 

The ultraviolet absorption spectrum of the coenzyme corresponds to that of uridine. Bromine destroys the uracil moiety of the coenzyme and effects the destruction of biological activity. 

The ribonucleotide reductase of Lactobacillus leichmanii catalyses the rapid conversion of enzyme-bound coenzyme B12 to an intermediate which has an absorption spectrum in the visible and u.v. like that of cob(ii)alamin (Biar)- Coffman et al. have recently analysed the e.s.r. spectrum of this intermediate and suggest that it contains low-spin cobalt(ii) with strongly distorted six-fold co-ordination. 

The second modified flavin of natural origin to be discovered was 8a-S-cysteinyl-FAD, the coenzyme of monoamino oxidase from liver and kidney outer mitochondrial membranes. Taking their departure from investigations of Yasunobu (8J) and Hellerman (SO), which indicated the presence of covalently bound flavin in preparations of this enzyme, Singer and his group (85, 185) isolated the flavinyl peptide by degradation of MAO with trypsin-chymotrypsin and identified cysteine as the amino acid residue bound next to the flavin moiety (184). The absorption spectrum of the flavin peptide from monoamino oxidase is readily differentiated from that of riboflavin by a hypsochromic shift of the second absorption band (360 nm, compare with 372 for riboflavin), in the neutral oxidized state (44, 184). It is similar to that of 8a-histidyl-riboflavin in the cationic state in that the band centered around 400 nm (abs. max. 375 nm, shoulder at 410 nm) is partially resolved. The fluorescence emission (4, 30) is only 10% of that of riboflavin, but oxidation with peracids raises it to 90% of riboflavin emission. 

The proton NMR spectrum of the coenzyme pyridoxal phosphate in 2HzO is shown in Fig. 3-24 as obtained with a 60-MHz spectrometer. Four things can be measured from such a spectrum (1) the intensity (area under the band). In a proton NMR spectrum, areas are usually proportional to the numbers of equivalent protons giving rise to absorption bands ... 

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