Spectrofluorimetric method for measuring the activity of the enzyme alpha-L-fucosidase using the ion associate of 2-chloro-4-nitro phenol-rhodamine-B.
A low cost and accurate method for the detection and analytical determination of the activity of the enzyme alpha-L-fucosidase (AFU) was developed. The method was based upon measuring the fluorescence intensity of the complex ion associate of the ion associate of rhodamine-B and the compound 2-chloro-4-nitrophenol (RB(+)CNP(-)) at 580 nm in phosphate buffer (pH 5) against the reagent blank. The influence of the different parameters, e.g. pH, incubation time, temperature, 2-chloro-4-nitrophenol concentration, foreign ions and surfactants that control the fluorescence intensity of the produced ion associate was critically investigated. The correlation between the fluorescence activity of the enzyme AFU by the developed procedures and the standard method was positive and highly significant in patients and controls (r(2)=0.99, p<0.001). The developed method is simple and proceeds without practical artifacts compared to the standard method.
El-Shahawi
M S
MS
Department of Chemistry, King Abduaziz University, Jeddah 21589, Saudi Arabia. mohammad_el_shahawi@yahoo.co.uk
Othman
A M
AM
El-Houseini
M E
ME
Nashed
B
B
Elsofy
M S
MS
D019694:Hepatitis B, Chronic / Q000097:blood / Q000201:enzymology; D019698:Hepatitis C, Chronic / Q000097:blood / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry / Q000378:metabolism*; D009596:Nitrophenols / Q000737:chemistry; D015203:Reproducibility of Results; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D005644:alpha-L-Fucosidase / Q000097:blood / Q000737:chemistry / Q000378:metabolism*
D007477:Ions; D009596:Nitrophenols; D012235:Rhodamines; D005644:alpha-L-Fucosidase; C029773:rhodamine B; C024836:4-nitrophenol