Dear community,
I am having this issue as I need to follow the following protocol:
"Phosphoglucoisomerase-glucose-6-phosphate dehydrogenase (PGI/G6PDH): 40 ul of phosphoglucose isomerase (Boehringer yeast enzyme, 2 mg/ml, ca. 350 units/mg) and 20 ul of glucose-6-phosphate dehydrogenase (Boehringer yeast enzyme, 5 mg/ml, ca. 140 units/mg) are diluted to 1 ml with 20 mM Tris-HC1 or imidazole-HCl"
I am confused about the 2 mg/ml, ca. 350 units/mg. Could someone help?
Thank you.
Best regards,
Katherine
2 mg/ml is the protein concentration.
250 units/mg is the specific activity of the enzyme.
Here, units refer to units of enzyme activity, typically expressed as micromoles of product formed/minute under a specific set of conditions.
Adding to Adam B Shapiro answer, you need to recognize that not all protein preparations are 100% pure and not all are 100% active. So 1mg/ml of some enzyme does not actually tell you how much enzyme activity you have. You need to calculate that from knowing the specific activity of that specific preparation of the enzyme.
Hi there,
In a coupled assay the essential issue is to introduce enzymes in sufficient amounts so that catalysis is considered instantaneous (for you it's conversion of Fructose-6-P into G6P then into 6P-gluconolactone in presence of NADP (generating NADPH)). With both enzyme concentration and enzyme specific activity you can calculate the number of units of each enzyme introduced in the reaction. For instance, in your protocol 28 units of PGI is introduced ( 0.04mL*(2mg/mL)*(350U/mg)) and 14 units of G6PDH (0.02mL*(5mg/mL)*(140U/mg)).
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