A rapid inhibitive assay for the determination of heavy metals using α-chymotrypsin; a serine protease
AbstractAgricultural areas in Malaysia depend upon unpolluted source of water and hence need continuous monitoring. An enzyme-based assay for the detection of xenobiotics is a promising low cost and rapid method for monitoring. An enzymatic-heavy metal assay has been developed using α-chymotrypsin; a serine protease. This enzyme is assayed by using casein as a substrate with Coomassie dye to track the complete hydrolysis of casein. In the absence of inhibitors, casein is hydrolysed to completion and the solution is brown. Eight heavy metals were tested on the α- chymotrypsin bioassays and in the presence of Hg2+, Cr2+ and Zn2+ at the final concentration of 5mg/L, hydrolysis of casein is inhibited and the solution remains blue. The assay was sensitive toHg2+, Cr2+ and Zn2+ with an IC50 (the concentration of toxicant giving 50% inhibition) of 1.34 mg/L, 1.974 mg/L and 2.49 mg/L, respectively. The calculated limits of quantitation (LOQ) value for Hg2+, Cr2+ and Zn2+ were 0.012 mg/L, 0.324 mg/L and 0.098 mg/L, respectively. The calculated LOD values for Hg2+, Cr2+ and Zn2+ were 0.003 mg/L, 0.320 mg/L and 0.074 mg/L, respectively. The results of this study indicate that this assay has the potential to be used as a tool in biosensor development for the determination of heavy metals in various samples.
Alina M, Azrina A, Mohd Yunus AS, Mohd Zakiuddin S,
Mohd Izuan Effendi H, Muhammad Rizal R. Heavy metals
(mercury, arsenic, cadmium, plumbum) in selected marine fish
and shellfish along the Straits of Malacca. Int Food Res J.
, 19(1): 135-140.
Liu JGRA, Goyer RA, Waalkes MP. Toxic effects of
metals. Casarett & Doull’s toxicology: the basic science of
poisons, 4th Ed. 1991, Hightstown, NJ. 931-979.
Sharma S, Raghuvanshi BPS, Shukla S. Toxic Effects of Lead
Exposure in Rats: Involvement of Oxidative Stress,
Genotoxic Effect, and the Beneficial Role of NAcetylcysteine
Supplemented with Selenium. J Environ Pathol
Tox On. 2014: 33; 19-32.
Shukor MY, Baharom NA, Rahman FA, Abdullah MPA,
Shamaan NA, Syed MA. Development of a heavy metals
enzymatic-based assay using papain. Anal Chim Acta. 2006;
Han SZ, Yuan ZM, Li X. Methylene blue-mediated enzyme
electrode for the determination of trace mercury (II), mercury
(I), methylmercury, and mercury-glutathione complex,
Biosens Bioelectron. 2001; 16: 9-16.
Jung K, Bitton G, Koopman B. Assessment of urease
inhibition assays for measuring toxicity of environmental
samples, Water Res. 1995; 29: 1929-1933.
Shukor MY, Masdor N, Baharom NA, Jamal JA, Abdullah
MPA, Shamaan NA, Syed MA (2008). An inhibitive
determination method for heavy metals using bromelain, a
cysteine protease. Appl Biochem Biotechnol. 2008; 144: 283-
Shukor MY, Baharom NA, Masdor NA, Abdullah MPA,
Shamaan NA, Jamal JA, Syed MA. The development of the
inhibitive determination for zinc using a serine protease. J
Environ Biol. 2009; 30: 17-22.
Obara, M, Karasaki Y, M Ohno. α-Chymotrypsin-catalyzed
hydrolysis of peptide substrates effect on the reactivity of the
secondary interaction due to the peptide moiety C-terminal to
the cleaved bond. J Biochem, 1979; 86: 461-468.
Bradford MM. A rapid and sensitive for the quantitation of
microgram quantities of protein utilizing the principle of
protein-dye binding, Anal Biochem. 1976; 72: 248-254.
Bickerstaff GF, Zhou H. Protease activity and autodigestion
(autolysis) assays using Coomassie blue dye binding. Anal
Biochem. 1993; 210: 155-158.
Peterson GL. A simplification of the protein assay method of
Lowry which is more generally applicable. Anal biochem,
; 83: 346-356.
Bensadoun A, Weinstein D, Steinberg D. Assay of proteins in
the presence of interfering material, Anal Biochem. 1976; 70:
Smith PK, Krohn, R. I., Hermanson, GT, Mallia AK, Gartner
FH, Provenzano M, Klenk DC. Measurement of protein
using bicinchoninic acid. Anal Biochem. 1985; 150: 76-85.
Burokerkilgore M, Wang KK. A Coomassie brilliant blue
G-250-based colorimetric assay for measuring activity of
calpain and other proteases. Anal Biochem. 1993; 208: 387-
Botsford JL. A simple assay for toxic chemicals using a
bacterial indicator. World J Microb Biot. 1998; 14: 369-376.
Miller JN, Miller JC, Statistics and Chemometrics for
Analytical Chemistry, 4th Ed., Prentice Hall, England, 2000,
Hsieh CY, Tsai MH, Ryan DK, Pancorbo OC. Toxicity of the
priority pollutant metals to Vibrio fisheri in the Microtox®
chronic toxicity test. Sci total environ. 2004; 320: 37-50.
Copyright (c) 2014 Nanobio and BioNano
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).