Specific protein analysis by light-scatter measurement with a miniature centrifugal fast analyzer.

The MB isoenzyme of creatine kinase (EC 2.7.3.2) may be prepared in vitro by dissociation of the MM and BB dimers into the M and B monomers. These monomers then recombine in a random manner to form all possible combinations-MM, MB, and BBthe presence of which may be detected by chromatography or electrophoresis. Dawson et al. (1) describe this process for animal tissues, while Keutel et a!. (2) prepared a MB hybrid isoenzyme from human and animal tissues. We prepared the MB isoenzyme by using a human serum with only MM in high activity and BB from either human brain or small intestine. The procedure was as follows. The MM serum, 2 ml, was combined with 2 ml of the BB-containing tissue extract. Sufficient urea to make the 4-ml mixture 8 molar was added directly and dissolved at room temperature with gentle mixing. The mixture was dialyzed vs. a buffer [0.1 mol each of tris(hydroxymethyl)aminomethane and 2-mercaptoethanol per liter, pH 7.5]. Dialysis was for two days at 4 01, with one change of the external fluid each day. The urea was determined occasionally, and dialysis was considered complete when the urea concentration had returned to that of the patient’s original serum or less. A hybrid isoenzyme with the electrophoretic and chromatographic characteristics of MB isoenzyme was found in the mixture where it had not existed before. In two such experiments 14% and 26% of the enzyme was the MB isoenzyme. This method of in vitro synthesis of the MB isoenzyme presents a relatively easy and convenient procedure for preparing a suitable CK isoenzyme control containing all three human isoenzymes. The stability of the three hybridized isoenzymes requires further study.