Spectrophotometric detection of bacteriolytic activity of diluted lysostaphin solutions |
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Authors: | I Márová J Kovář |
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Institution: | (1) Laboratory of Skin Biochemistry, 1st Department of Dermatovenerology, Medical Faculty, Masaryk University, 656 91 Brno, Czech Republic |
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Abstract: | Turbidimetric method with spectrophotometric detection of changes in density of test bacteriaS. aureus strain SA 812 for determination of bacteriolytic activity of lysostaphin was employed. Results of two evaluations are compared:
(1) calculation of the relative value of turbidity decrease on the basis of the difference of absolute values ofA
540 at the beginning of reaction and after the incubation period, (2) following of time changes inA
540 by monitoring the course of reaction directly in the constant-temperature cuvette of the spectrophotometer at 37°C. Both
arrangements yielded identical results, within the significance level of 0.05. With concentrated samples both methods yield
reliable results; with diluted samples the accuracy of the “absolute” method decreases together with decreasing lysostaphin
concentration much faster than with the “registration” method. The registration method makes it possible to detect even minute
amounts of the lytic enzyme and thus to distinguish the values of activity in dilute samples even when data obtained by means
of the “absolute” method cannot be considered as reliable. A unit of bacteriolytic activity can be expressed from the kinetic
curve as an amount of enzyme preparation causing ΔA
540/min=0.01. |
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Keywords: | |
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