Yong, Lee Shin
(2013)
Effect of buffer composition on the background activities
of choline kinase.
Project Report.
Pusat Pengajian Sains Kesihatan, Universiti Sains Malaysia.
(Submitted)
Abstract
Choline kinase has become one of the popular enzymes that has recently been
studied extensively as it functions to catalyze the phosphorylation of choline to form
phosphocholine in the presence of ATP and magnesium. Choline kinase exists in
three isoforms which are CKal, CKa2 and CKp encoded by two separate genes,
Chka and Chkfl. Pyruvate kinase-lactate dehydrogenase (PK.-LDH) coupled
enzymatic assay was used to determine the activities for hCKa2 and hCKp.
Background activity was the activity observed before choline was added into the
reaction. The existence of background activity in enzymatic assay of hCKa2 and
hCK.p affects the accuracy of measurement for choline kinase. Thus, different buffer
systems at pH 8.5 were used to compare the effect of these buffers towards the
background activity of hCKa2 and hCKp. The aim of this study was to reduce the
background activity of choline kinase by using different buffer systems (pH 8.5) for
enzymatic assay. The buffers tested for enzymatic assay were borate, glycine-NaOH,
HEPES, Tris-acetate, Tris-maleate and tricine. Kinetic parameters for hCKa2 and
hCKp were determined with buffers that removed the background activity of the
enzymes. The buffers that abolished the background activity of choline kinase were glycine-NaOH, HEPES and tricine buffers. The highest Vmax for hCKa2 of 87.41
pmol/min/mg with choline as the substrate was obtained in tricine buffer, while HEPES buffer gave the highest Vmax of 41.60 pmol/min/mg for hCKp. With ethanolamine as substrate, the highest Vmax for both hCKa2 and hCKp were obtained
in tricine buffer with the values of 37.71 and 13.86 pmol/min/mg respectively. In
conclusion, glycine-NaOH, HEPES and tricine buffers were able to remove the
background activities of the hCKa2 and hCKp. The activity level of these enzymes
were also dependent on the buffer system used in enzymatic assay.
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