Department of Medicine Combined Locked Nucleic Acid and Molecular Beacon Technologies for Sensitive Detection of the Jak2 Somatic Single-base Sequence Variant

polymerization, a large change in conformation, or a combination of both (2, 3). The control Bence Jones protein had 2 molecular species that were consistent with monomer and dimer in the presence or absence of physiologic concentrations of salt. Sedimentation equilibrium experiments were performed on the cryo -light chain protein that had been preequilibrated in the no-salt buffer and run at 6000 and 12 000 rpm at 20 °C (analysis could not be performed at 4 °C because of protein precipitation at higher concentrations). Sedimentation equilibrium centrifugation analysis with a selfassociation model revealed that the mean molecular mass of the cryo protein in the absence of salt at 20 °C was Mr 60 000. The sedimentation data were consistent with a mixture of 70% dimers and 30% tetramers. Early studies investigating the mechanisms of temperatureand concentration-dependent cryoprecipitation have shown that low temperatures change the intramolecular environment of the protein, particularly with certain aromatic residues (4 ). It is possible that there are both saltand temperature-dependent changes at 20 °C and that only the salt-dependent changes in polymerization are detected by either sedimentation equilibrium or by the apparent Mr measured by gel filtration at 4 °C. Low salt accurately reflects the presence of trimers and hexamers that would form a cryoprecipitate at the higher concentration of light chain protein found in undiluted urine stored at 4 °C. The sodium concentration in urine can vary considerably, and we did not ascertain the sodium in the urine of the cryo patient. It is noteworthy, however, that the control light chain did not show any salt-dependent changes in relative molecular mass, unlike the cryo protein. It is likely that several physicochemical factors contributed to urine cryoglobulin formation in this patient, including the immunoglobulin light chain protein sequence, resulting from the inherent diversity in the variable regions of immunoglobulins, and the concentration, because the protein had to be diluted substantially to prevent precipitation on the gel-filtration column at 4 °C. In addition, there was no evidence of clinical symptoms related to the urine cryo at physiologic temperature. The urine remained a clear liquid at room temperature and 37 °C and showed complete gel formation only at 4 °C. However, because we do not have data to confirm the role of these various factors, the extent to which these may have contributed to cryo formation in this patient remains speculative.