Sodium polyanethol sulfonate (SPS) falsifies protein staining and quantification and how to solve this problem.

Sodium polyanethol sulfonate (SPS) is an anionic detergent with a broad range of activities and applications. While studying the excretion of cytoplasmic proteins in Staphylococcus aureus SPS was used as cell lysis inhibitor. When investigating the protein pattern of culture supernatants from cells grown in the absence or presence of SPS by Coomassie blue stained polyacrylamide gel the amount of protein bands was significantly decreased in the presence of SPS, suggesting that this effect was due to inhibition of cell lysis. However, various control studies showed that the apparent decreased protein secretion was an artifact due to the interference of SPS with Coomassie blue- and silver-staining. The only alternative method that was uninfluenced by SPS was imidazole-SDS-zinc staining. This is the method of choice particularly when protein interfering compounds are present in the extracts. For protein quantification in liquid samples the bicinchoninic acid (BCA) assay appeared to be the method of choice in the presence of SPS. The assay is based on neutral peptide bonds and is therefore rather insensitive to interfering compounds. This study shows that SPS and most likely also related detergents might falsify conventional protein staining and quantification methods.

Vascular endothelial cell killing by combinations of membrane-active agents and hydrogen peroxide.

Previous studies have demonstrated that a number of membrane-active agents are capable of binding to the surface of polymorphonuclear leukocytes (PMN) resulting in an augmentation of superoxide anion and hydrogen peroxide (H2O2) production in response to soluble stimuli. It is now demonstrated that these same membrane-active agents can bind to the surface of endothelial cells and enhance their susceptibility to killing by H2O2. Membrane-active agents which are capable of synergizing with H2O2 include cationic proteins, cationic poly-amino acids, lysophosphatides and enzymes which are capable of degrading membrane phospholipids (e.g., phospholipase C, phospholipase A2 and streptolysin S). In each case, treatment of the target cells with the membrane-active agent and H2O2 produces greater damage than the sum of the damage produced by either agent separately. Since inflammatory lesions, particularly sites of bacterial infection, may contain a rich mixture of cationic substances, phospholipases and phospholipid breakdown products, these substances may contribute to the tissue damage observed at sites of inflammation by enhancing endothelial cell sensitivity to PMN-generated H2O2 as well as by augmenting the generation of H2O2 by PMNs.

Inactivation of the polyanionic detergent sodium polyanetholsulfonate by hemoglobin.

Sodium polyanetholsulfonate (SPS) has been added to blood culture media for many years. Its incorporation results in a higher yield of positive blood cultures due to its inactivation of antimicrobial cationic compounds. The most active of these cations include complement components, aminoglycoside-aminocyclitol antibiotics, and receptors on polymorphonuclear leukocytes. There have been reports from studies conducted outside patient blood culture bottles that SPS itself may possess antibacterial activity against some isolates of Neisseria meningitidis, Neisseria gonorrhoeae, and Peptostreptococcus anaerobius. Conversely, in patient clinical trials there has been no significant difference in pathogen isolation rates in the presence or absence of SPS. In an attempt to explain this in vitro/in vivo disparity, a search was undertaken to elucidate which variable constituent in blood, heretofore not studied quantitatively, might have a major effect on modulating the activity of SPS. It was found that hemoglobin combined stoichiometrically with SPS with a Kd of approximately 10(-7) mol/liter. Optimum SPS inactivation occurred at an SPS/hemoglobin ratio of 1:6 (wt/wt). SPS-sensitive isolates of N. gonorrhoeae and N. meningitidis were protected by the addition of hemoglobin from the antimicrobial effects of this polyanion in time-kill studies. This protection was directly related to the amount of SPS combined in solution. Therefore, the amount of free hemoglobin in solution must be measured when studying the antimicrobial activity of polyanions or when evaluating the effect of different polyanions on the recovery rates of pathogens in patient blood culture clinical trials.

Potentiation of anchorage-independent colony formation by sodium polyanethol sulphonate.

Sodium polyanethol sulphonate (SPS) when incorporated into rat erythrocyte lysate (REL) containing semi-solid agar medium at 1 mg ml-1. markedly enhanced colony formation by a number of anchorage-independent cell lines. REL usually needed to be included for the expression of SPS induced potentiation as in its absence SPS was generally cytotoxic. Studies suggested that SPS reduced the lag prior to colony initiation resulting in the earlier appearance of colonies and in a higher cloning efficiency. The effectiveness of SPS in potentiating colony formation by responsive cell lines was markedly influenced by the species of serum and to a lesser extent by differences between individual batches. Enhancement by SPS was greater with poorer foetal calf serum (FCS) batches than with better. This effect may have been partly due to SPS interfering with the action of a growth inhibiting serum component, possibly a lipoprotein. Studies in which delipidated FCS was substituted for normal FCS suggested that SPS was also able to compensate for the lack of a growth-promoting lipid component. Binding studies showed that initially 125I-SPS bound equally well at 4 degrees C and 37 degrees C with continued labelling occurring only at 37 degrees C. Autoradiography of cells labelled at 37 degrees C for 24 h revealed the presence of intracytoplasmic 125I-SPS.

Staphylococcus aureus requires increased level of Ca(2+) or Mn(2+) to grow normally in a high-NaCl/low-Mg(2+) medium.

Mg2+-availability in Staphylococcus aureus cells decreased significantly with increasing NaCl concentration in growth media. Cells grew in a NaCl-free, Chelex resin-treated complex medium only if the medium was supplemented with 50 microM MgCl2, while, growth was limited when the medium was further supplemented with 1.0 M NaCl. Cells grown in such a high-NaCl/low-Mg2+ medium exhibited the morphologic abnormality of larger than normal cells. Both sufficient growth and normal cell morphology were restored by increasing Mg2+ concentration in a high-NaCl medium, or by supplementation with either CaCl2 or MnSO4 in a high-NaCl/low-Mg2+ medium. Supplementing with BaCl2, SrCl2 or FeSO4, however, had no effect. These results indicate that Ca2+ and Mn2+ might play some essential role in the growth of Staphylococcus aureus in a high-NaCl/low-Mg2+ environment.