SERS, cytochrome

Fatty acyl CoA may be elongated and desaturated (to a limited extent in humans) using enzymes associated with the smooth endoplasmic reticulum (SER). Cytochrome is involved in the desaturation reactions. These enzymes carmot introduce double bonds past position 9 in the fetty add. 

Principle After the test substance has been demethylated by the microsomal enzyme system of the SER (cytochrome P 450), the labelled carbon atoms are catabolized to C02. The velocity of demethylation can be measured by determining the C02 value in expired air. Catabolization of aminopyrine is enhanced with elevated activity of the cytochrome P 450 and reduced accordingly due to the loss of liver cell volume. The metabolization is independent of perfusion. Exposure to radiation is minimal. 

Figures 3.91(a) and (b) show cyclic voltammograms of the SERS electrode in aqueous solutions of the SSBipy and PySH. In the potential range —0.3 V to 0.3 V vs. SCE, which is the range of interest for the reversible reduction of cytochrome c, no notable faradaic currents were observed for either of these species. However, at potentials < —0.4 V SSBipy is reduced to PySH and the PySH so formed is re-oxidised to SSBipy at potentials >0.1 V. Similarly, PySH is oxidised to SSBipy at potentials >0.1 V and this product re-reduced at potentials < —0.4 V.

Figure 3.92 shows SERS spectra of adsorbed SSBipy and PySH at 0 V in the absence of the solution species, together with the Raman spectra of PySH in solution and crystalline SSBipy. The activities of the modified electrodes were first confirmed in solution containing cytochrome c. 

In addition to the effect of mutations at Phe-82 on the stability of the cytochrome c active site, the intense, negative Soret Cotton effect in the circular dichroism spectrum of ferricytochrome c is profoundly affected by the presence of non-aromatic amino acid residues at this position [115]. Recent examination of six position-82 iso-l-ferricytochrome c mutants establishes that while Tyr-82 exhibits a Soret CD spectrum closely similar to that of the wild-type protein, the intensity of the negative Soret Cotton affect varies with the identity of the residue at this position in the order Phe > Tyr > Gly > Ser = Ala > Leu > He, though the Ser, Ala, He, and Leu variants have effectively no negative Soret Cotton effect [108]. 

Steady state kinetics and protein-protein binding measurements have also been reported for the interaction of these mutant cytochromes with bovine heart cytochrome c oxidase [120]. The binding of cytochrome c variants to the oxidase occurred with increasing values of Kj in the order He (3 x 10 Mol L ) < Leu = Gly < wild-type < Tyr < Ser (3 x 10 molL ). Steady-state kinetic analysis indicated that the rate of electron transfer with cytochrome c oxidase increased in the order Ser < He < Gly < Leu < Tyr < wild-type, an order notably different from that observed for a related analysis of the oxidation of these mutants by cytochrome c peroxidase [85]. This difference in order of mutant turnover by the oxidase and peroxidase may arise from differences in the mode of interaction of the cytochrome with these two enzymes. 

Fig. 2a-c. Stereodiagram of the yeast iso-1-cytochrome c surface, (a) Surface of the wild-type protein (b) surface of the Ser-82 mutant (c) surface of the Gly-82 mutant. (Modified from Refs. [123, 124])... 

In the liver s hepatocytes, the proportion represented by the sER is particularly high. It contains enzymes that catalyze so-called biotransformations. These are reactions in which apolar foreign substances, as well as endogenous substances—e. g., steroid hormones—are chemically altered in order to inactivate them and/or prepare them for conjugation with polar substances (phase I reactions see p. 316). Numerous cytochrome P450 enzymes are involved in these conversions (see p. 318) and can therefore be regarded as the major molecules of the sER. 

Cytochromes P-450 may be found in other organelles as well as the SER including the rough endoplasmic reticulum and nuclear membrane. In the adrenal gland, it is also found in the mitochondria, although here adrenodoxin and adrenodoxin reductase are additional requirements in the overall system. Although the liver has the highest concentration of the enzyme, cytochromes P-450 are found in most, if not all, tissues. 

The most important enzyme involved in bio transformation is cytochrome P-450, which catalyzes many phase 1 reactions. This enzyme is located primarily in the SER (microsomal fraction) of the cell and is especially abundant in liver cells. Cytochrome P-450 primarily catalyzes oxidation reactions and consists of many isoforms (isozymes). These isoenzymes have overlapping substrate specificities. The most important subfamily in humans is CYP3A4, although there is considerable variation in CYP3A4 expression between individuals. 

The primary structure of a protein is the sequence of its amino acids. For example, the first 10 amino acids in the cytochrome c sequence are Ala-Ser-Phe-Ser-Glu-Ala-Pro-Gly-Asn-Pro, while the first 10 amino acids in the myosin sequence are Phe-Ser-Asp-Pro-Asp-Phe-Gln-Tyr-Leu-Ala. Therefore, the primary structure is just the full sequence of amino acids in the polypeptide chain or chains. Finding the primary structure of a protein is called protein sequencing. The first protein to be sequenced was the hormone insulin. 

Table VII summarizes the conditions for chymotryptic hydrolysis of the proteins and peptides listed in Table VI. The parameters which would be expected to determine the rate of hydrolysis (apart from the nature of the bonds in the particular substrates) are temperature, pH, time of hydrolysis, and the molar ratio of chymotrypsin to substrate. All these factors often differ considerably for the substrates listed. Hydrolyses have been performed under conditions which vary from 2 to 24 hr, from pH 7.0 to 9.0, from 22° to 40°C, and at enzyme to substrate molar ratios between 1/360 to 1/21. It is not obvious how variations in pH and temperature affect the apparent specificity of chymotrypsin, but at low molar ratios of enzyme to substrate only the most susceptible bonds would be expected to be hydrolyzed. The lowest molar ratio was employed in the studies with ribonuclease. The only bonds of an unusual nature which were split were those formed by serine and histidine in the following sequences, -Thr-Ser. . . Ala-Ala- and -Lys-His. . . Ileu-Ileu-. Many of the unusual splits listed in Table VI were observed in equine or human cytochrome c and in oxidized papain. Each of these substrates was digested for long periods of time and at high ratios of enzyme to substrate under conditions which would be expected to split bonds that are usually resistant to hydrolysis.

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