The extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted of subunits containing heme groups, monomers and trimers, and nonheme structures, called linkers, and the whole protein has a minimum molecular mass near 3.1 x 10(6) Da. This and other proteins of the same family are useful model systems for developing blood substitutes due to their extracellular nature, large size, and resistance to oxidation. HbGp samples were studied by dynamic light scattering (DLS). In the pH range 6.0-8.0, HbGp is stable and has a monodisperse size distribution with a z-average hydrodynamic diameter (D(h)) of 27 +/- 1 nm. A more alkaline pH induced an irreversible dissociation process, resulting in a smaller D(h) of 10 +/- 1 nm. The decrease in D(h) suggests a complete hemoglobin dissociation. Gel filtration chromatography was used to show unequivocally the oligomeric dissociation observed at alkaline pH. At pH 9.0, the dissociation kinetics is slow, taking a minimum of 24 h to be completed. Dissociation rate constants progressively increase at higher pH, becoming, at pH 10.5, not detectable by DLS. Protein temperature stability was also pH-dependent. Melting curves for HbGp showed oligomeric dissociation and protein denaturation as a function of pH. Dissociation temperatures were lower at higher pH. Kinetic studies were also performed using ultraviolet-visible absorption at the Soret band. Optical absorption monitors the hemoglobin autoxidation while DLS gives information regarding particle size changes in the process of protein dissociation. Absorption was analyzed at different pH values in the range 9.0-9.8 and at two temperatures, 25 degrees C and 38 degrees C. At 25 degrees C, for pH 9.0 and 9.3, the kinetics monitored by ultraviolet-visible absorption presents a monoexponential behavior, whereas for pH 9.6 and 9.8, a biexponential behavior was observed, consistent with heme heterogeneity at more alkaline pH. The kinetics at 38 degrees C is faster than that at 25 degrees C and is biexponential in the whole pH range. DLS dissociation rates are faster than the autoxidation dissociation rates at 25 degrees C. Autoxidation and dissociation processes are intimately related, so that oligomeric protein dissociation promotes the increase of autoxidation rate and vice versa. The effect of dissociation is to change the kinetic character of the autoxidation of hemes from monoexponential to biexponential, whereas the reverse change is not as effective. This work shows that DLS can be used to follow, quantitatively and in real time, the kinetics of changes in the oligomerization of biologic complex supramolecular systems. Such information is relevant for the development of mimetic systems to be used as blood substitutes.

This work reports the use of two-dimensional (2D) liquid chromatography system coupled with a tandem mass spectrometry for the quantification of ampicillin in bovine milk. A restrict access media column (RAM-BSA C(8) , 50 × 2.1 mm, Luna, 10 μm, 100 Å) was used in the first dimension in order to exclude macromolecules, while an ACQUITY UPLC BEH C(18) (50 × 2.1 mm, 1.7 μm) column was used in the second dimension. Three different channels of selected reaction monitoring (SRM) were used: 350 > 106 m/z, 350 > 160 m/z, and 350 > 192 m/z. The first transition was used for the quantification (higher intensity), and latter two for confirmation. The developed method is simple and requires a total analysis time of only 14 min/sample. The sample treatment involved only a centrifugation step for 20 min. The validated method has been successfully applied to monitor AMP residues in raw milk samples. To our knowledge, this is the first study to report the use of ultrahigh-performance liquid chromatography (UHPLC) in 2D configuration.