Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

Abstract

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.

Electrophoretic Analysis of Serum Proteins in Cystic Fibrosis

“…It has been realized for some time that urea increases the pK values of dissociable groups and the measured pH of aqueous solutions, presumably by reducing the activity of hydrogen ions (12), and the fact that urea also increases the apparent pH of carrier ampholyte solutions and thus the measured pI in isoelectric focusing has been discussed by several investigators ( 2 3 , 3 5 , 4 0 , 44,45). Although the degree of elevation of pH will depend on the urea concentration and the buffering system, our studies and the reports in the literature ( Figures 1, 2 , and 3 of this rcport, our measured pI for this protein, using three different pH gradients, agrees well with the expected valucs based on reports in the literature (25,28,(31)(32)(33)(34)(35). Concerning the most cationic proteins in serum, the true pI's of these protcins havc not bcen realized, primarily because of the heretofore persistent problems in alkaline range isoelectric focusing in polyacrylamide gels (43,48).…”

“…We base this conclusion on the pI's of a specific marker protein (sperm whale myoglobin) and-the most cationic serum proteins (those with the highest PI'S). Several investigations on isoelect~ic focusing of sperm whale myoglobin have demonstrated a pI of 8.1 + 0.1 for the major component of this protein in the ferric state (25,28,(31)(32)(33)(34)(35). We also have obtained this value for our preparation of sperm whale myoglobin when it was focused in the absence of urea.…”

Isolation and Characterisation of Glycoproteins from Sputum

“…Solution of mucus gels in 6 M urea is believed to depend on the solubilisation of hydrogen bonds [45] but on prolonged treatment it is possible that mechanical shearing of bonds due to stirring may also contribute to the solubilisation. It has recently been suggested [46] that 6 M urea results in intramolecular disulphide interchange in bovine plasma albumin on prolonged treatment. Our results indicate that there is a correlation between ease of solubility of the mucus gels and the protein content of the component glycoproteins, the gels containing glycoproteins of higher protein content being less soluble in 6 M urea.…”

The Use of Analytical Isoelectric Focusing for Detection and Identification of -Lactamases

“…This was carried out on sheets of polyacrylamide gel (2 I ' 5 x I 6.5 x 0.1 5 cm) supported on glass plates (Awdeh, Williamson & Askonas, 1968 ;Salaman & Williamson, 1971). The composition of the gel mixture was as follows: acrylamide, 70 mg/ml ; N,N'-methylenebisacrylamide, 2 mg/ml ; N,N,N',N'-tetramethylethylenediamine, 0.25 ,ug/ml; riboflavin, 0.002 mg/ml; pH 3 5 to 10 ampholine, 20 mg/ml.…”