BackgroundThe positive antimicrobial effects of increasing concentrations of thiocyanate (SCN-) and H2O2 on the human peroxidase defence system are well known. However, little is known about the quantitative efficacy of the human peroxidase thiocyanate H2O2 system regarding Streptococcus mutans and sanguinis, as well as Candida albicans. The aim of this study was to evaluate the effect of the enzyme lactoperoxidase on the bactericidal and fungicidal effectiveness of a thiocyanate-H2O2 combination above the physiological saliva level. To evaluate the optimal effectiveness curve, the exposure times were restricted to 1, 3, 5, and 15 min.ResultsThe bactericidal and fungicidal effects of lactoperoxidase on Streptococcus mutans and sanguinis and Candida albicans were evaluated by using two test mixtures of a 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide solution, one without and one with lactoperoxidase. Following the quantitative suspension tests (EN 1040 and EN 1275), the growth of surviving bacteria and fungi in a nutrient broth was measured. The reduction factor in the suspension test without lactoperoxidase enzyme was < 1 for all three tested organisms. Thus, the mixtures of 2.0% (w/v; 0.34 M) thiocyanate and 0.4% (w/v; 0.12 M) hydrogen peroxide had no in vitro antimicrobial effect on Streptococcus mutans and sanguinis or Candida albicans. However, the suspension test with lactoperoxidase showed a high bactericidal and fungicidal effectiveness in vitro.ConclusionThe tested thiocyanate and H2O2 mixtures showed no relevant antimicrobial effect. However, by adding lactoperoxidase enzyme, the mixtures became not only an effective bactericidal (Streptococcus mutans and sanguinis) but also a fungicidal (Candida albicans) agent.
The reaction of ascorbate with recombinant hemoglobin (rHb1.1) in the presence of differing partial pressures of oxygen was studied. In the presence of 15 000 ppm (1.5%) residual oxygen, ascorbate/oxygen-mediated reactions resulted in an increased rate of autoxidation, modification of the beta-globin, increased oxygen affinity and decreased maximum Hill coefficient. One of the observed modifications to the beta-globin was a 72 Da addition to its N-terminus. Detailed characterization indicates the modification was an imidazolidinone type structure. Thorough deoxygenation of the hemoglobin solution to <150 ppm of oxygen prior to addition of ascorbate was required to prevent these modifications. Addition of ascorbate to the deoxy hemoglobin (deoxyHb) at pH 8 induced aggregation, eventually leading to precipitation. No such precipitation was observed at pH 7. Long-term storage of the hemoglobin was carried out by addition of ascorbate to deoxyHb at pH 7. The level of methemoglobin remained at <2% for up to 1 year at 4 degreesC, with no detectable precipitation of the protein. Modifications similar to those observed by the acute studies were observed over the 1-year period and correlated with disappearance of the added ascorbate.
We have prepared a dry film for the enzymic determination of total serum cholesterol. It consists of a transparent support bearing a buffered gelatin layer, and a white reflective spreading layer that contains all of the necessary components for the detection of cholesterol. The method is based on (a) hydrolysis of cholesterol esters to cholesterol by cholesterol ester hydrolase (EC 3.1.1.13), (b) oxidation of cholesterol to cholest-4-en-4-one and hydrogen peroxide by cholesterol oxidase (EC 1.1.3.6), and (c) oxidation of a triarylimidazole leuco dye with hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) to produce a dye with maximum absorption at about 650 nm. For use over a wider range of concentration, the dye density is read at 540 nm. With reflection densitometry and appropriate mathematical transformation, readings and cholesterol concentrations are linearly related to 5500 mg/L. Results correlate well with those by the Abell-Kendall comparison method (slope 0.97, intercept 92.5, correlation coefficient 0.974, Sy.x = 250.7), and the method is precise (CV of 1.2-2.3% for a control fluid and patients' samples) and relatively free of interferences.
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