title: Stability of HAMLET--a kinetically trapped alpha-lactalbumin oleic acid complex.
abstract: The stability toward thermal and urea denaturation was measured for HAMLET (human alpha-lactalbumin made lethal to tumor cells) and alpha-lactalbumin, using circular dichroism and fluorescence spectroscopy as well as differential scanning calorimetry. Under all conditions examined, HAMLET appears to have the same or lower stability than alpha-lactalbumin. The largest difference is seen for thermal denaturation of the calcium free (apo) forms, where the temperature at the transition midpoint is 15 degrees C lower for apo HAMLET than for apo alpha-lactalbumin. The difference becomes progressively smaller as the calcium concentration increases. Denaturation of HAMLET was found to be irreversible. Samples of HAMLET that have been renatured after denaturation have lost the specific biological activity toward tumor cells. Three lines of evidence indicate that HAMLET is a kinetic trap: (1) It has lower stability than alpha-lactalbumin, although it is a complex of alpha-lactalbumin and oleic acid; (2) its denaturation is irreversible and HAMLET is lost after denaturation; (3) formation of HAMLET requires a specific conversion protocol.
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Cristol Building, Room 226, Boulder, CO 80309-0215, USA. jfast@colorado.edu
authors.forename: Jonas
authors.initials: J
authors.lastname: Fast
authors.forename: Ann-Kristin
authors.initials: AK
authors.lastname: Mossberg
authors.forename: Catharina
authors.initials: C
authors.lastname: Svanborg
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D001059:Apoproteins / Q000737:chemistry*; D017209:Apoptosis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002417:Cattle; D002470:Cell Survival; D002621:Chemistry / Q000379:methods; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004587:Electrophoresis, Agar Gel; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008892:Milk / Q000378:metabolism; D008894:Milk Proteins / Q000737:chemistry; D008956:Models, Chemical; D019301:Oleic Acid / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D040901:Proteomics / Q000379:methods; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014466:Ultraviolet Rays; D014508:Urea / Q000494:pharmacology
chemical_list: D001059:Apoproteins; D008894:Milk Proteins; C041966:apo-alpha-lactalbumin; D019301:Oleic Acid; D014508:Urea; D007768:Lactalbumin; D000645:Ammonium Sulfate; D002118:Calcium