Following irradiation, samples were analysed by SDS PAGE using a 5% stacking gel and 15% resolving gel under denaturing conditions. Lane 1: molecular weight Selleckchem Natural Product Library marker, lane 2: L-S-, lane 3: L-S+, lane 4: L+S- (1.93 J/cm2), lane 5: L+S- (3.86 J/cm2), lane 6: L+S- (9.65 J/cm2), lane 7: L+S+ (1.93 J/cm2), lane 8: L+S+ (3.86 J/cm2), lane 9: L+S+ (9.65 J/cm2). L = samples

exposed to laser light and S = samples exposed to 20 μM methylene blue. The apparent molecular mass of the V8 protease was approximately 30 kDa. α-haemolysin Table 1 shows the effect of photosensitisation of α-haemolysin with 1, 5, 10 and 20 μM methylene blue and laser light. Concentrations of 5, 10 and 20 μM methylene blue completely

inhibited the haemolytic activity of the enzyme when exposed to laser light (L+); DNA Damage inhibitor therefore inactivation of the toxin occurs even see more at photosensitiser doses that are sub-inhibitory to EMRSA-16 (i.e. 5 μM). There was no effect on the activity when the enzyme was incubated with the methylene blue in the absence of laser light (L-). To investigate the effect of light dose on the activity of α-haemolysin, the enzyme was exposed to 20 μM methylene blue and irradiated with 665 nm laser light for 1, 2 and 5 minutes. Table 2 shows that the activity of the enzyme was completely inhibited after exposure to a light dose of 1.93 J/cm2 in the presence of 20 μM methylene blue, and further investigation showed that a laser light dose as low as 0.64 J/cm2 results in the complete inhibition of haemolytic activity

when treated with 20 μM methylene blue (data not shown). Laser light alone had no appreciable effect on the activity of the α-haemolysin. SDS PAGE analysis (Figure 6) showed that bands derived from the α-haemolysin after photosensitisation with 20 μM methylene blue and laser light became less well defined and smeared with increasing irradiation time compared to untreated samples. This result is similar to that observed for the V8 protease. The addition of 12.5% human serum did not affect the ability of photosensitisation to inactivate the α-haemolysin, and complete inhibition of haemolytic Tyrosine-protein kinase BLK activity was observed after treatment of the toxin with 20 μM methylene blue and a laser light dose of 1.93 J/cm2 in the presence of serum. This finding is consistent with the inactivation of the toxin in the absence of serum. Table 1 The effect of treatment of α-haemolysin with different concentrations of methylene blue and a laser light dose of 1.93 J/cm2. Concentration of methylene blue (μM) Haemolytic titre L- Haemolytic titre L+ 1 1/1024 1/256 5 1/1024 1/2 10 1/1024 < 1/2 20 1/512 1/2 An equal volume of either 1, 5, 10 and 20 μM methylene blue or PBS was added to S. aureus α-haemolysin and samples were either exposed to 1.93 J/cm2 laser light (L+) or kept in the dark (L-).