Full title- A COMPARISON OF THE INHIBITORY EFFECTS OF METAL IONS
Enzymes are an essential part for the functioning of the human system. Most of the enzymes require binding of metal ions and other non-protein components for their appropriate functioning. Such non proteins, which facilitate the functioning of enzymes, are called cofactors. The cofactors can be an organic cofactor as Thymine Pyrophosphate (TPP), Flavin Adenine Dinucleotide (FAD), Nicotinamide Adenine dinucleotide (NAD); or inorganic cofactors like metal ions, Mg2+, Cu2+, Mn2+, Zn2+ and Iron-sulphur clusters. When the enzyme is bound to a cofactor, ready to carry out its function, it is known as a ‘Holoenzyme.' An enzyme without a co-factor is called an ‘Apoenzyme.' Simple enzymes require one or two cofactors for their functioning, while complex enzymes require several cofactors for their functioning. Accordingly, human system requires the intake of metals in small quantities called ‘trace elements’ for proper functioning of its integral enzyme components, and hence proper metabolism.
Environment is the place of occurrence of metal ions. Also, the trace elements required for proper functioning of the enzymes are heavy metals.
Heavy metals have high atomic weight and are almost five times dense than water. Now- a- days, with the technological advancement use of heavy metals have increased at a dramatic rate in all sectors like agriculture, household, industries. Increased use of heavy metals is raising serious concerns regarding exposure of humans and plants to toxic doses of heavy metals. Risk factors associated with heavy metal toxicity involve age, gender, exposure route and nutritional status of the person exposed. Cadmium, Chromium, Mercury, Arsenic are amongst considered being most toxic and, hence, having importance in public health.
The focus of this lab report is heavy metal toxicity. We have studied the activity of an enzyme, called β-galactosidase (Jacobson et al. 1994), in response to heavy metal toxicity. Other names of is β-galactosidase are beta/β gal. This enzyme carries out hydrolysis reaction of galactosides to convert them into simple sugars such as monosaccharides. Various complex carbohydrates like glycoproteins, ganglioside GM1, lactose, lactosylceramides are the substrates of beta/β gal. There are various methods to estimate the quantity of β-galactosidase present in the mammalian system (Garry and Kindell, 2005).
The active site of beta/β gal is responsible for the enzyme's hydrolysis reaction via "Shallow" and "Deep" binding mechanisms. Substrate binding of the enzyme beta/β gal is facilitated by the presence of divalent magnesium (Mg2+) and monovalent potassium (K+) ions. However, there takes place a competitive inhibitory binding of toxic metal ions like copper, zinc, cadmium and mercury. Binding of toxic metal ions to the enzyme beta/β gal results in impaired functioning of the enzyme. It is primarily by the ingestion of heavy metals present in soil or environment which results in heavy metal toxicity (Smith, 2009). There are several in vitro tests available for the estimation of heavy metal toxicity (Apartin and Ronco, 2001).In this report, we present the data from metal toxicity on beta galactosidase enzyme assessed by simple fluorometric analysis. Enzyme was treated with different concentrations of four heavy metals, Copper, Zinc, Cadmium and Mercury, along with the substrate of the enzymes.
RESULTS
Toxicity of copper on beta gal enzyme
The results obtained in quadruplets were analysed to obtain the enzyme activity, in response to, copper toxicity. Values were normalized to Time 0 for each plate and average fluroimeteric values of all plates are tabulated as
Enzyme activity was calculated as per the formula
%age activity = Test value (i.e. in presence of metal)-Blank X 100
Control value (in absence of metal) -Blank
The results obtained for percentage activity are tabulated as
Also, percent inhibitions of the enzyme beta gal, in response to, each concentration of copper were calculated by subtracting the percent activity from 100. The results are tabulated as
The IC 50 obtained was 1.67 ppm of copper ion. The results for percent activity are graphically represented as
The results of percent inhibition of beta gal, in response to, different concentrations of copper represented graphically as
Toxicity of Zinc on beta gal enzyme
The results obtained in quadruplets were analysed to obtain the enzyme activity, in response to, copper toxicity.
Values were normalized to Time 0 for each plate and average fluroimeteric values of all plates are tabulated as
Enzyme activity was calculated as per the formula
%age activity = Test value (i.e. in presence of metal)-Blank X 100
Control value (in absence of metal) -Blank
The results obtained for percentage activity are tabulated as
The IC 50 obtained was 1.50 ppm of Zinc ion. The results for percent activity are graphically represented as
Also, percent inhibitions of the enzyme beta gal, in response to, each concentration of zinc were calculated by subtracting the percent activity from 100. The results are tabulated as
The results of percent inhibition of beta gal, in response to, different concentrations of zinc represented graphically as
The negative value of percent inhibition was plotted as per the raw data, in which the enzyme activity was reported to be higher than the control value. This essentially means that the enzyme activity was more than 100% upon exposure to low concentrations of Zinc. The enzyme activity of the controls (no metal present) was taken as 100%. Alternatively, the percent inhibition of enzyme in control samples was taken as 0%.
Toxicity of Cadmium on beta gal enzyme
The results obtained in quadruplets were analysed to obtain the enzyme activity, in response to, copper toxicity.
Values were normalized to Time 0 for each plate and average fluroimeteric values of all plates are tabulated as
Enzyme activity was calculated as per the formula
%age activity = Test value (i.e. in presence of metal)-Blank X 100
Control value (in absence of metal) -Blank
The results obtained for percentage activity are tabulated as
The IC 50 obtained was 1.20 ppm of Cd ion. The results for percent activity are graphically represented
Also, percent inhibitions of the enzyme beta gal, in response to, each concentration of Cadmium were calculated by subtracting the percent activity from 100. The results are tabulated as
The results of percent inhibition of beta gal, in response to, different concentrations of cadmium represented graphically as
The negative value of percent inhibition was plotted as per the raw data, in which the enzyme activity was reported to be higher than the control value. This essentially means that the enzyme activity was more than 100% upon exposure to low concentrations of Cadmium. The enzyme activity of the controls (no metal present) was taken as 100%. Alternatively, the percent inhibition of enzyme in control samples was taken as 0%.
Toxicity of Mercury on beta gal enzyme
The results obtained in quadruplets were analysed to obtain the enzyme activity, in response to, copper toxicity.
Values were normalized to Time 0 for each plate and average fluroimeteric values of all plates are tabulated as
Enzyme activity was calculated as per the formula
%age activity = Test value (i.e. in presence of metal)-Blank X 100
Control value (in absence of metal) -Blank
The results obtained for percentage activity are tabulated as
The IC 50 obtained was extrapolated as 0.2 ppm of Hg ion. The results for percent activity are graphically represented
Also, percent inhibitions of the enzyme beta gal, in response to, each concentration of Mercury were calculated by subtracting the percent activity from 100. The results are tabulated as
The results of percent inhibition of beta gal, in response to, different concentrations of mercury represented graphically as
Relative toxicities of the four heavy metal ions tested
Toxicity of any metal ion is inversely correlated to the IC50 dosage. In other words, lower IC50 corresponds to a high toxic metal. In contrast, a metal having a higher IC50 confers lower toxicity to the biological system. A graphical representation of relative toxicities of Copper, Zinc, Cadmium and Mercury was plotted as
DISCUSSION AND CONCLUSION
Mercury and Cadmium are amongst the top listed heavy metals, which pose serious health risks to humans, plants and marine life (Tchounwou et al. 2012). In accordance with that, our results also indicate higher toxicity levels of Mercury and Cadmium to the enzyme beta galactosidase, in contrast to the toxicity exhibited by Copper and Zinc. Mercury is a highly toxic pollutant and toxicant. Exposure to mercury causes gastro, neuro and nephro- toxicity.
All four metals conferred their respective toxicities, and hence, reduced activity of the β galactosidase enzyme was observed. The IC 50 of Copper and Zinc showed similar values of 1.5-1.6 ppm. However, the IC 50 of Cadmium was 1.2 ppm. Interestingly, mercury exhibited an IC 50 of 0.2 ppm. We can conclude that lower value of IC 50 corresponds to a high metal toxicity. In other words, mercury was able to give rise to a 50% inhibition of β galactosidase activity only at a concentration of 0.2 ppm. So, from our experimental data it is evident that the mercury is most toxic followed by Cadmium, and Zinc and Copper.
References
- Jacobson, R. H.; Zhang, X. -J.; Dubose, R. F.; Matthews, B. W. (1994). Three-dimensional structure of β-galactosidase from E. Coli. Nature 369 (6483): 761–766.doi:10.1038/369761a
- Gary RK, Kindell SM (2005). Quantitative assay of senescence-associated beta-galactosidase activity in mammalian cell extracts. Anal. Biochem. 343 (2): 329–34.doi:10.1016/j.ab.2005.06.003. PMID 1600495
- Apartin C & Ronco A (2001). Development of a free beta-galactosidase in vitro test for the assessment of heavy metal toxicity. Environmental Toxicology,16(2):117-20.
- Smith SR (2009). A critical review of the bioavailability and impacts of heavy metals in municipal solid waste composts compared to sewage sludge. Environmental International, 35(1):142-56. doi: 10.1016/j.envint.2008.06.009.
- Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ, (2012). Heavy metal toxicity and the environment. Molecular, Clinical and Environmental Toxicology Experientia Supplementum ;101:133-64. doi: 10.1007/978-3-7643-8340-4_6.
