Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds.

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and L-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25000 vs. approximately 85000). A sensitivity of 75 ng (approximately 500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

Determination of molecular-mass distribution of food-grade protein hydrolyzates by size-exclusion chromatography and chemiluminescent nitrogen detection.

Size-exclusion chromatography (SEC) and chemiluminescent nitrogen detection (CLND) were used to estimate the molecular-mass distribution of food-grade protein hydrolyzates. Simultaneous CLND and UV (214 nm) detection is demonstrated for analytical SEC of an experimental casein hydrolyzate. In order to validate the estimated average M(r) values derived from the SEC column data, a preparative SEC separation of an extensive casein hydrolyzate was pursued. Fractions were collected on a time basis and analyzed by time-of-flight (TOF) mass spectrometry. A plot of TOF M(r) vs. SEC M(r) indicated that the peptides below M(r) of 1200 were eluted as estimated by the calibrated preparative SEC column. This paper demonstrates the power of using a dual CLND and UV detection system for analytical SEC analysis of protein hydrolyzates with a calibrated column.

Optimization of Chemiluminescent Nitrogen Detection for Packed-Column Supercritical Fluid Chromatography with Methanol-Modified CO(2).

A chemiluminescent nitrogen detector was optimized for packed-column supercritical fluid chromatography. Methanol modifier concentrations of 15% or less, an ozone flow of 5.8 mL/min, and a decompressed CO(2) flow between 240 and 310 mL/min were found to exhibit a maximum sensitivity of 5 ng for sulfamethazine (1 ng of N). The addition of a membrane drier to the "optimized" system further decreased the minimum detectable quantity (MDQ) to 0.5 ng (0.1 ng of N). In addition, by using a microbore column (2 mm i.d.) instead of an analytical scale column, the postcolumn decompressed flow split could be eliminated, further reducing the MDQ to 0.125 ng of sulfamethazine (0.025 ng of N).

Chemiluminescent nitrogen detection as a new technique for purity assessment of synthetic peptides separated by reversed-phase HPLC.

Reversed phase high-performance liquid chromatography with chemiluminescent nitrogen detection (HPLC-CLND) was used for the quantitative analysis of peptides manufactured by solid-phase peptide synthesis. CLND provided quantitative information regarding the nitrogen distribution of peptide samples following HPLC separation. This technique permits the universal quantitation of the "peptide content" of synthetic peptides in an on-line mode without pre- or post-column derivatization and free of interference from non-nitrogen-containing UV chromophores. This paper will show the utility of this novel technique in measuring the peptide content of crude synthetic proinsulin chain C peptide. A mixture of five reference peptides was analyzed to show the homogeneity of the CLND response and used to determine peptide content. Application for the purified product is also discussed. The detection profiles were acquired in parallel with a UV detector.

1H NMR study of the influence of hydrophobic contacts on protein-prosthetic group recognition in bovine and rat ferricytochrome b5.

The proton nuclear magnetic resonance spectra of the soluble fragment of native bovine and genetically engineered wild-type rat ferricytochrome b5 reconstituted with a wide variety of hemes chemically modified at 2- and/or 4-positions have been recorded and analyzed. While all but one nonsymmetric heme yielded comparable amounts of the two heme orientations immediately after reconstitution, the relative proportion of the two orientations at equilibrium varied widely. The unpaired spin density distribution in the heme pi system leads to substituent hyperfine shift patterns in these paramagnetic complexes that are completely diagnostic of the heme orientation in the protein matrix. An empirical assignment strategy is outlined and applied which allows unequivocal assignment of the absolute orientation of a derivatized heme within the protein matrix. Using a series of hemes lacking 2-fold symmetry solely due to a single substitution, the preferences for localized site occupation of vinyls, methyls, and hydrogens are developed. The large differences in relative stability of the two orientations of native protohemin in the two cytochromes b5 is shown to result from the additivity of localized effects for the bovine protein and the near cancellation of competing effects in the rat protein. The major determinant of the heme orientation is judged to be a repulsive interaction between a vinyl and a hydrophobic cluster of amino acids including positions 23 and 25. The differences in this heme orientational preference among bovine, rat, and chicken ferricytochromes b5 could be correlated with the relative steric bulk of the residues at positions 23 and 25.(ABSTRACT TRUNCATED AT 250 WORDS)

13C-NMR study of labeled vinyl groups in paramagnetic myoglobin derivatives.

The 13C-NMR spectra of high-spin met-aquo myoglobin, spin-equilibrium met-azido myoglobin, low-spin met-cyano myoglobin, deoxy myoglobin and carbonmonoxy myoglobin from sperm whale reconstituted with hemin 13C enriched at both vinyl alpha or beta positions have been recorded. In all cases the labeled vinyl 13C signals are clearly resolved and useful spectra could be obtained within approx. 15 minutes. The decoupling of multiplet structure due to attached proton(s) has led to the specific assignment of vinyl 13C alpha signals in all paramagnetic derivatives and the 13C beta signals in met-cyano myoglobin. In all other cases, the collapse of the proton multiplet structure as a function of 1H decoupling frequency has located, but not assigned, the attached 1H resonance positions which are obscured by the intense diamagnetic envelope in the 1H-NMR spectrum. The resulting vinyl 13C hyperfine shifts follow Curie behavior, and the patterns closely resemble those in the appropriate model complexes in the same oxidation/spin/ligation state, except that the protein exhibits more in-plane asymmetry. The hyperfine shift patterns are indicative of dominant pi contact shifts for all ferric complexes. Deoxy myoglobin vinyl 13C and 1H contact shifts provide little evidence for pi bonding.