LEU2 enzyme assay

cathelijne van Duren C.M.J.Van-Duren at herts.ac.uk
Tue Nov 3 14:01:52 EST 1998


>
>Dear all,
>
>Enzyme assay is killing me!!!!!!!!!!!!!
>
>
>I am trying to do an enzymatic assay for the inducibility of the LEU2 gene
>(B-isopropyl malate) according to Parsons and Burns paper,Metods in
>enzymology 1970, 108, p793-799 .
>
>The reaction is as follows:
>B-isoprpylmalate + NAD -----> a-Ketoisocaproate + NADH + CO2 There are two
>ways of measuring;
>A	NADH spectrophotometrically at 340 nm
>B	a-Ketoisocaproate colimetrically as the 2,4-dinitrophenylhydrazone
>
>
>My problem with this assay is that I have a very low absorbance using any
>of the two methods. The curve reaches its max at 0.1 OD. It is also
>observed that there is a high background. without the subtrate present
>there is already a conversion of NAD to NADH. I span my crude cell extracts
>at 13000 rpm for 25 min on a bench top microcentrifuge to remove the
>oxidase, but with very little succes. It was suggested to me so increase
>the NAD conc. from 0.01M to 1M. The conc of the substrate is not limiting
>the reaction so I really could do with some suggestions here. I have given
>the details of the procedure below sothat may there is someone out there
>who has tried this protocol and can help me out.
>
>Thanks for your help,
>
>
>Kate
>C.M.J.van-duren at herts.ac.uk
>
>
>
>A	Reagents used:
>0.3 ml potassium phosphate 1M pH=8
>0.15 ml KCl 1M
>0.15 ml MgCl2 0.01M
>0.2 ml b-Isopropylmalic acid 0.01M (substrate) 0.2 ml NAD 0.01M
>enzyme diluted in 0.05M potassium phosphate pH=7 SDW to make up to 3 ml
>reaction volume
>
>Look at initial rates spectrophotometrically, 25C
>
>
>
>
>B	Reagents used:
>0.3 ml potassium phosphate 1M pH=8
>0.1 ml KCl 1M
>0.1 ml MgCl2 0.01M
>0.1 ml b-Isopropylmalic acid 0.01M (substrate) 0.2 ml NAD 0.01M
>enzyme diluted in 0.05M potassium phosphate pH=7 SDW to make up to 2 ml
>reaction volume
>
>Assay mixture minus enzyme is equilabrated at 37C for 4 min, add enzyme,
>start reaction for XX min stop reaction: add 3ml 2,4-dinitrophenylhydrazine
>		incubate 15 min ar RT
>		add 1ml 40% KOH
>read absorbace at 540nm
>








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