A continuous spectrophotometric assay method for peptidylarginine deiminase type 4 activity.
A simple, continuous spectrophotometric assay for peptidylarginine deiminase (PAD) is described. Deimination of peptidylarginine results in the formation of peptidylcitrulline and ammonia. The ammonia released during peptidylarginine hydrolysis is coupled to the glutamate-dehydrogenase-catalyzed reductive amination of alpha-ketoglutarate to glutamate and reduced nicotinamide adenine dinucleotide (NADH) oxidation. The disappearance of absorbance at 340nm due to NADH oxidation is continuously measured. The specific activity obtained by this new protocol for highly purified human PAD is comparable to that obtained by a commonly used colorimetric procedure, which measures the ureido group of peptidylcitrulline by coupling with diacetyl monoxime. The present continuous spectrophotometric method is highly sensitive and accurate and is thus suitable for enzyme kinetic analysis of PAD. The Ca(2+) concentration for half-maximal activity of PAD obtained by this method is comparable to that previously obtained by the colorimetric procedure.
Liao
Ya-Fan
YF
Department of Life Sciences, National Chung-Hsing University, Taichung 402, Taiwan, ROC.
Hsieh
Hui-Chieh
HC
Liu
Guang-Yaw
GY
Hung
Hui-Chih
HC
D002021:Buffers; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002623:Chemistry Techniques, Analytical; D003124:Colorimetry; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D006867:Hydrolases / Q000032:analysis* / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D011994:Recombinant Proteins / Q000032:analysis / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D002021:Buffers; D011994:Recombinant Proteins; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases; D008274:Magnesium; D002118:Calcium