Characterization of the particles of purified kappa-casein: trypsin as a probe of surface-accessible residues.

kappa-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogeneous polymers, however, self-associate into nearly spherical particles with an average diameter of 13 nm at pH 8.0, as revealed by negatively stained transmission electron micrographs and dynamic light scattering. The weight-average molecular weight of the aggregates at pH 8.0, as judged by analytical ultracentrifugation, is 648,000. Trypsin digestion at pH 8.0 was used to probe the surface groups of the kappa-casein A polymers. The reaction with trypsin was rapid and the peptides liberated were identified by separation with reverse-phase HPLC, amino acid analysis, and protein sequencing. The most rapidly released peptides (t1/2 < 30 sec) were from cleavage at Arg 97 and Lys residues 111 and 112. These results suggest a surface orientation for these residues, and the data are in accord with earlier proposed 3D predictive models for kappa-casein. It is speculated that Arg 97, together with adjacent His residues (98 and 100) and Lys residues 111 and 112, form two positively charged clusters on the surface of the otherwise negatively charged casein. These clusters bracket the neutral chymosin cleavage site (whose hydrolysis triggers a well-known digestive process) and so these clusters may facilitate docking of the substrate caseins with chymosin.

Rapid electrodialytic clean-up of biological samples for high-performance liquid chromatography.

A sample clean-up system employing electrodialysis with size-selective and charge-selective membranes is described. When applied to the treatment of 0.5-ml milk samples containing sulfamethazine, the system produced an undiluted, clear solution in 3 min and eliminated the components in untreated milk that caused column fouling and double peaks. In contrast to conventional liquid- and solid-phase extraction procedures, electrodialytic clean-up is readily automated and uses no organic solvents.

Reactions of hydroxyamino acids during hydrochloric acid hydrolysis.

Chlorosubstitution reactions occur readily during HCl hydrolysis of delta- and epsilon-hydroxynorleucines (Hnle), the products of deamination of poly-L-lysine by nitrite at low pH. During amino acid analysis, chloronorleucines elute as new peaks after delta- and epsilon-Hnle. To determine if other hydroxyamino acids undergo similar changes during hydrolysis, they were subjected individually to HCl hydrolysis conditions with and without added phenol. Amino acid analyses indicated that terminal hydroxy groups on linear side chains undergo reactions during HCl hydrolysis; the products appear as new peaks which may be chloroderivatives. In contrast, no new peaks are observed in HCl hydrolysates of delta-hydroxylysine or amino acids with beta-hydroxy groups (beta-hydroxynorvaline, serine, and threonine). Phenol did not protect linear amino acids from reactions during HCl hydrolysis but did prevent loss of the cyclic amino acids tyrosine, hydroxyproline, and 3,4-dihydroxyphenylalanine. Although the gamma-hydroxy group of homoserine would be expected to undergo reaction, HCl catalyzes its cyclization to form homoserine lactone instead.

Gas chromatographic/mass spectrometric determination of carbon isotope composition in unpurified samples: methamphetamine example.

A gas chromatograph/quadrupole mass spectrometer system, operated in electron impact/selected ion monitoring mode, is used to determine the intensity ratio of the m/z 59 and the m/z 58 ions of the [C3H8N]+ fragment derived from methamphetamine samples synthesized with varying amounts of 13C-labeled methylamine. Crude products are introduced into the gas chromatograph without prior cleanup. The ratios measured were in excellent agreement with those calculated. A change in 0.25% use of 13C-methylamine is sufficient for product differentiation. The feasibility of using isotope labeling and subsequent mass spectrometric isotope ratio measurement as the basis of a compound tracing mechanism is discussed. Specifically, if methamphetamine samples manufactured from legal sources are asked to incorporate distinct 13C compositions, their sources can be traced when samples are diverted into illegal channels. Samples derived from illicit preparations can also be traced if the manufacturers of a precursor (methylamine in this case) incorporate distinct 13C compositions in their products.