Small amounts of urea and guanidine hydrochloride can be detected by a far-UV spectrophotometric method in dialysed protein solutions.
The quantization of small amounts of chemical denaturants as urea or guanidine hydrochloride in protein solutions after dialysis is a difficult task in the molecular biology laboratory practice. Refractometric methods are useful to quantify a denaturant in the molar range but this methodology is not helpful when the denaturant is present in small amounts. The method herein described is a new comparative method that requires, a priori, the quantification of the stock solutions of urea (8 M) and guanidine hydrochloride (6 M) by refractometry to prepare by sequential dilution the standards used for comparison in the spectropolarimeter. The method is based on the observation that the wavelengths, at which the absorbance of polarized light increases in the far-UV region, as observed by spectropolarimetry, is related to the concentration of the chemical denaturant present in the protein solution. In the quantitation method herein reported, the urea and guanidine hydrochloride detection limits range from 1.2 x 10(-4) to 6 x 10(-6) M depending on the protein dialysis buffer used for a standard cell path length of 1 cm. The sensibility of this method results to be comprised in a range 4-5 orders of magnitude higher than that measured by refractometry. The determinations in both the sample and the control preparations are virtually completed within approximately 10 min.
Fuertes
Miguel Angel
MA
Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma de Madrid, 28049 Madrid, Spain. mafuertes@cbm.uam.es
Pérez
José Manuel
JM
Alonso
Carlos
C
D045424:Complex Mixtures / Q000032:analysis; D015314:Dialysis Solutions / Q000032:analysis* / Q000592:standards; D019791:Guanidine / Q000032:analysis* / Q000592:standards; D008832:Microchemistry / Q000379:methods* / Q000592:standards; D011506:Proteins / Q000032:analysis*; D012016:Reference Values; D012031:Refractometry / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods* / Q000592:standards; D014508:Urea / Q000032:analysis* / Q000592:standards
D045424:Complex Mixtures; D015314:Dialysis Solutions; D011506:Proteins; D014508:Urea; D019791:Guanidine