Thermal and Alkaline Denaturation of Bovine  -Casein

Abstract The precision of replicate analyses for alkaline phosphataseactivity measured on a survey reference sample was extremely poor. The reference sample's enzyme itself became suspect and was demonstrated to be sensitive to alkaline denaturation-in sharp contrast to the stability of the alkaline phosphatasesfound in human serum. The stability and chemical reactivity of the phosphatases present in this reference sample and in pooled frozen human serum, as well as those found in 4 partially purified nonhuman preparations and 4 commercial serum control materials, were investigated with regard to heat and alkaline denaturation, electrophoretic migration, and inhibition by phosphate, EDTA, and L-pheflylalanine. It was concluded that criteria of stability and chemical reactivity, as well as more detailed information concerning the source of enzymes utilized in reference samples and control materials, are needed. On the basis of these studies, reliance on commercial serum enzyme control materials as an enzyme "standard" cannot be endorsed.

Download Full-text

Accuracy of the haemoglobin alkaline denaturation test for detecting maternal blood contamination of fetal blood samples for prenatal karyotyping

Prenatal Diagnosis ◽

10.1002/(sici)1097-0223(199910)19:10<927::aid-pd668>3.0.co;2-h ◽

1999 ◽

Vol 19 (10) ◽

pp. 927-929 ◽

Cited By ~ 7

Author(s):

Waldo Sepulveda ◽

Cecilia Be ◽

Ronald Youlton ◽

Jorge Gutierrez ◽

Eduardo Carstens

Keyword(s):

Maternal Blood ◽

Fetal Blood ◽

Blood Contamination ◽

Blood Samples ◽

Alkaline Denaturation

Download Full-text

Alkaline denaturation and partial refolding of pepsin investigated with DAPI as an extrinsic probe

Biophysical Chemistry ◽

10.1016/s0301-4622(97)00016-1 ◽

1997 ◽

Vol 67 (1-3) ◽

pp. 75-83 ◽

Cited By ~ 16

Author(s):

Roberto Favilla ◽

Alessandra Parisoli ◽

Alberto Mazzini

Keyword(s):

Alkaline Denaturation

Download Full-text

Structural dissection of alkaline-denatured pepsin

Spectroscopy An International Journal ◽

10.1155/2004/769354 ◽

2004 ◽

Vol 18 (2) ◽

pp. 227-236 ◽

Cited By ~ 1

Author(s):

Yuji O. Kamatari ◽

Christopher M. Dobson ◽

Takashi Konno

Keyword(s):

Limited Proteolysis ◽

Helical Structure ◽

Cd Spectroscopy ◽

Core Region ◽

Detailed Knowledge ◽

Native State ◽

Denatured State ◽

Residual Structure ◽

Alkaline Denaturation ◽

Folded Core

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IPstate) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IPstate in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IPstate of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IPstate is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.

Download Full-text