Prosthetic groups, measurements

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

In assays of enzyme activities a cofactor, but not a prosthetic group, can be easily lost from the enzyme by dilution during extraction or purification, or removed by agents that will bind the cofactor. For these reasons, an excess of cofactor is routinely added to the assay medium (e.g. in kinase assays) for the measurement of enzyme activity. 

A Ca +-binding protein isolated from jellyfish Aequorea sp.) that is frequently used as a chemiluminescent calcium indicator.Aequorin contains a hydrophobic prosthetic group, coelenterazine. Investigators have been able to express the transfected gene recombinantly in a number of cells, and upon addition of the cofactor, one can measure intracellular calcium concentration. See Fura-2... 

The electronic properties of haemoproteins have been measured and discussed in recent years by workers whose primary interests cover a wide range of scientific disciplines, from theoretical physics to medicine and biology. In fact there can be few other fields in which so many disciplines have pooled their resources, both experimental and theoretical. In spite of the prodigious development of other physical methods electronic absorption spectroscopy remains the most widely-used tool in the study of these proteins. A proper understanding of their spectra is clearly of the greatest importance in the investigation of the molecular electronic structure of the haem chromophore, and of the effects of the structure and conformation of the polypeptide chain on the properties of the prosthetic groups derived from it. 

Heme prosthetic groups have strong absorption in visible-Soret spectral regions. This heme absorption is extremely sensitive to the stmcture of porphyrin as well as to the redox and ligand binding states of the iron. Thus, hemes a and as must exhibit differences in their absorption spectra even if their respeetive iron atoms are in the same oxidation state, since the number of ligands bound to heme a is different from the number of ligands bound to heme as. Careful measurements of the absorption spectra and the... 

The measurement by Simmons ei al. (1961) of a far ultraviolet Cotton effect associated with helical polypeptides has already been cited in connection with the rotatory properties of the peptide bond (see Section II, D). Another set of investigations has focused on the optical activity which a symmetrical group, either a side chain, prosthetic group, or dye, assumes as a result of its interaction with an asymmetric protein or polypeptide. 

EPR signals for both the flavosemiquinone radical and the low-spin ferric heme have been reported (65, 78-82). The flavosemiquinone signal, which is easily observed at 123 K, shows a typical g value of 2.0039 0.002 (65). The bandwidth, which is around 15 G, is very like that of an anionic, or red, semiquinone (65). The EPR signal of the low-spin ferric heme can be observed at low temperatures ( 28 K) and shows g values of 2.99, 2.22, and 1,47 (65), which are similar to those found for cytochrome 65 (81). EPR rapid freezing studies have allowed the amounts of semiquinone and ferric heme to be monitored during reduction of the enzyme by L-lactate (66). This has proved to be extremely useful in the development of kinetic schemes to describe the flow of electrons in the enzyme. The distance between the prosthetic groups in H. anomala flavocytochrome 62 has been estimated from EPR experiments and spin-lattice relaxation measurements (82). Pyruvate was used to stablize the flavosemiquinone and the effect on the signal of this species from oxidized and reduced heme was measured. The results indicated a minimum intercenter distance of 18-20 A (82). 

Table 2.2 Thermodynamic and structural parameters of soluble proteins. Thermal denaturation free energy change, AG, under reducing conditions and comparable temperatures for an exhaustive set of monomeric uncomplexed proteins with disulfide bonds and without prosthetic groups or ion coordination [11]. Deviations from the balance relation are measured by Y-(5X + 20) and shown to anticorrelate tightly (R2 = 0.72, Fig. 2.3) with the denaturation free energies...

In enzyme-substrate reactions, radicals may be formed either on the enzyme (on a prosthetic group or the polypeptide), the substrate, or both. Enzyme-derived radicals tend to be immobilized and, depending on the experimental conditions, can be long lived. In contrast, substrate-derived radicals generally are tumbling rapidly in solution and are transient. Therefore, the methods used to characterize and quantitate the two kinds of radical tend to be quite different enzyme radicals are harder to identify with certainty but quantitation may simply require measuring a radical concentration rather than a rate of radical formation. 

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