Equine cytochrome

Bushey, M.M., Jorgenson, J.W. (1990a). A comparison of tryptic digests of bovine and equine cytochrome c by comprehensive reversed-phase HPLC-CE. J. Microcolumn Separations 2, 293-299. 

The minimum amount of analyte required for analysis depends strongly (as always) on the nature of the substance and on the properties of the ion source. Less than 100 zeptomole of tryptic peptides from bovine semm albumin has been detected by coupling nanoflow LC to an ESI source [264] and 0.4 nM of equine cytochrome c by employing an RF-field focusing funnel to improve transmission [265]. 

Similar behavior has been observed with equine cytochrome c as an oxidant. Thus, for the three above mentioned flavodoxins, the measured rate constants are 1.1 X 10 M s , 2.3 X 10 s and 1.7x10 s for the normal proteins... 

Recent studies on the electron-transfer between a number of flavin analogs with equine cytochrome c shows that flavin semiquinone reduction of cytochrome c reflects a diffusion-controlled process with a second-order rate of 6x 10 sec" ... 

Purves, R.W., Ells, B., Barnett, D.A., Guevremont, R., Combining H-D exchange and ESI-FAIMS-MS for detecting gas-phase conformers of equine cytochrome c. Can. J. Chem. 2005, 83, 1961. 

Figure 2.24. DESI mass spectrum of equine cytochrome c. (Reproduced from ref. 91 by permission of Wiley-Interscience, copyright 2005.)...

Other examples documenting the direct interaction between hydrophobic regions accessible on the surface of a protein and the corresponding nonpolar ligates on the stationary phase of a RPC or HIC sorbent have been described in the scientific literature. For example, as noted earlier, the hydrophobic contact site of equine cytochrome c and porcine growth hormone in the presence of /i-butyl ligands has been delineated [140,141] using a combination of footprint enzymatic procedures and electrospray mass spectrometry. A less direct approach is based... 

FIGURE 7.15 RPLC separation of tryptic peptide fragments of equine cytochrome c (peaks 1—10) on Chromolith SpeedROD RP-18e (a) 100 mm and (b) 50 mm. Source Reprinted from Ref. [66] with permission. 

BA, bioavailability BP, blood pressure CEE, conjugated equine estrogens CrCI, creatinine clearance CYP450, cytochrome P-450 DR, delayed-release Ml, myocardial infarction PV, per vagina Sign., significantly TD, transdermal TE, thromboembolism. 

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.

Figures 4.8 and 4.9 show the mass spectra of three proteins acquired on a MALDl-TOF andESl-QlT mass spectrometer, respectively. Both MALDl and ESI figures are of (a) cytochrome-c (equine), = 12,360.1, (b) apomyoglobin...

FiGURE 4.9 ESI ion trap mass spectra of three proteins (a) cytochrome-c (equine),... 

With the instrument tuned as described above, a set of test experiments were carried out to check the accuracy and consistency of mobility measurements made using the MoQToF. The experimental cross sections of the proteins cytochrome ubiq-uitin,(3 3 ) and lysozyme have been widely reported in the literature and are now frequently used to calibrate Synapt FIDS data. As these compounds are readily available they make ideal test/calibration compounds. Lyophilized, equine heart cytochrome c, ubiquitin, and hen s egg lysozyme were obtained from Sigma Aldrich and used without further purification. Samples were prepared for nano-ESl (electrospray ionization) in 50 50 methanol/water with 0.1% formic acid at concentrations ranging from 20 to 40 pM. 

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