Abstract: From 10 to >200 mg L-1 of protein has been determined in a flow injection apparatus with a detector cell containing a nickel oxide working electrode, a platinum counter-electrode and a SCE; the background electrolyte was 0.1 mM NiSO4 in 0.1 M NaOH, and amperometric measurements were made at 0.5 V. For denatured proteins, the detection limit was 1 mg l-1, and the technique has been used to study the denaturation kinetics of cytochrome c and serum albumin.

Abstract: A 0.018 cm2 Pt-disc electrode was immersed into phosphate buffer of pH 6.8 containing 200 mM pyrrole and as a mediator 100 mM ferricyanide ion (I), and a polypyrrole-I layer formed using a current density of 3 mA/cm2 at 850 mV vs. Ag/AgCl. Portions (20 µL) of cytochrome c (II) were injected into a carrier stream (1 ml/min) of 100 mM phosphate buffer of pH 10, and II was detected at 200 mV. Calibration graphs were non-linear up to 25 µM-II with a detection limit of 0.1 µM.

Abstract: The development and utilization of a Prussian blue/conducting polymer composite electrode was investigated. The introduction of a layer of conducting polymer (polypyrrole) enhanced the electroactivity of the outer Prussian blue film. This was used for improved signal generation for the redox protein cytochrome c. The effect of the thickness of these 2 film coatings and buffer solution pH was investigated. The anal. utility was demonstrated using flow injection anal.

Abstract: Pulsed hydrogen exchange/mass spectrometry, a new method for studying protein folding, has been used to investigate folding of cytochrome c on the 5 ms to 15 s time scale. Cytochrome c, unfolded in guanidine hydrochloride/D2O, was allowed to refold in a high-speed quenched-flow apparatus and pulse-labeled with protium to identify unfolded regions. Intact, labeled cytochrome c was digested into fragments which were analyzed by HPLC electrospray ionization mass spectrometry to determine the level of deuterium in each fragment. Bimodal distributions of deuterium were found for most segments, indicating that regions represented by these segments were either unfolded or completely folded in the intact polypeptide prior to labeling. This behavior is consistent with cooperative, localized folding which occurs in less than 10 ms in individual molecules. Deuterium levels found in the fragments were normalized to levels found in the same fragments derived from folded cytochrome c, pulse-labeled in the same manner, to indicate the percentage of cytochrome c that was folded. These results show that the N/C-terminal regions fold cooperatively on a time scale extending from less than the mixing time of the apparatus (5 ms) to as long as 15 s, and that the other regions also fold cooperatively. However, these regions do not begin to fold until 30 ms after mixing. In addition to providing new information on cytochrome c folding, these results demonstrate that pulse-hydrogen exchange/mass spectrometry is complementary to NMR in some respects and advantageous in others. Results of this study form the foundation required to extend the pulsed hydrogen exchange approach to folding studies of proteins too large to be analyzed by NMR.