Bicarbonate assay and phosphoenolpyruvate carboxylase enzyme from hyphomicrobium

Purified Phosphoenolpyruvate carboxylase (PEPC), wherein said PEPC has no more than 50% loss of activity after 60 days in 167 mM Tricine/HCL aqueous buffer, pH 8.0, and 15 mM MgCl.sub.2, at 25.degree. C., in the absence of stabilizer.

Purified phosphoenolpyruvate carboxylase (PEPC) having at least 2 of the characteristics selected from the group consisting of (i) at pH 6.5, an activity of at least 40% relative to its activity at pH 8.0 in 150 mM Tricine buffer, (ii) at pH 6.5, an activity of at least 50% relative to its activity at pH 8.0 in 150 mM Tricine buffer, (iii) a specific activity of at least 40 U/mg protein, and (iv) having no more than 50% loss of activity after 60 days in 167 mM Tricine/HCL aqueous buffer, pH 8.0, and 15 mM MgCl.sub.2, at 25.degree. C., in the absence of stabilizer.

An assay for measuring bicarbonate in a biological sample, comprising the steps of:

• (a) mixing said biological sample with a composition comprising PEPC which has an activity at pH 6.5 of at least 40% of its activity at pH 8.0 in 150 mM Tricine buffer, PEP, malate dehydrogenase, the reduced form of nicotinamide adenine dinucleotide (NADH) and a buffer, at a pH at or below 7;
• and
• (b) measuring the change in absorbance of light corresponding to the disappearance of NADH.

In an assay for measuring bicarbonate in biological samples under carboxylation conditions at or below pH 7, wherein the rate of the carboxylation reaction of phosphoenolpyruvate in the presence of PEPC is directly or indirectly measured and related to the bicarbonate concentration in the sample,

• the improvement wherein a PEPC is used which has an activity at pH 6.5 of at least 40% of its activity at pH 8,0 in 150 mM Tricine buffer.

An assay for measuring bicarbonate in a biological sample, comprising the steps of:

• (a) mixing said biological sample with a composition comprising PEPC which has a specific activity of at least 40 U/rag protein, PEP, malate dehydrogenase, NADH and a buffer;
• and
• (b) measuring the change in absorbance of light corresponding to the disappearance of NADH.

An assay for measuring bicarbonate in a biological sample, comprising the steps of:

• (a) mixing said biological sample with a composition comprising (i) PEPC characterized by no more than 50% loss of activity after 60 days in 167 mM Tricine/HCl aqueous buffer, pH 8.0, and 15 mM MgCl.sub.2, at 25.degree. C., in the absence of stabilizer, (ii) PEP, (iii) malate dehydrogenase, (iv) NADH and (v) a buffer;
• and
• (b) measuring the change in absorbance of light corresponding to the disappearance of NADH.

In an assay for measuring bicarbonate in biological samples under carboxylation conditions, wherein the rate of the carboxylation reaction of phosphoenolpyruvate in the presence of PEPC is directly or indirectly measured and related to the bicarbonate concentration in the sample,

• the improvement wherein a PEPC is used which has an activity of at least 40 U/mg protein.

In an assay for measuring bicarbonate in biological samples under carboxylation conditions, wherein the rate of the carboxylation reaction of phosphoenolpyruvate in the presence of PEPC is directly or indirectly measured and related to the bicarbonate concentration in the sample,

• the improvement wherein a PEPC is used which is characterized by no more than 50% loss of activity after 60 days in 167 mM Tricine/HCL aqueous buffer, pH 8.0, and 15 mMMgCl.sub.2, at 25.degree. C. in the absence of stabilizer.