Purification, microheterogeneity, and stability of human lipid transfer protein.
A method for the purification of lipid transfer protein (LTP) from human plasma was developed with the aid of succinylated low density lipoprotein-Sepharose affinity column chromatography. The purified LTP exhibited a single main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, upon isoelectric focusing on polyacrylamide gel, the preparations consistently showed nine bands with isoelectric points ranging from 4.6 to 5.4. The treatment of LTP with Clostridium perfringens neuraminidase shifted these multiple bands toward higher pH regions due to the release of sialic acid. Extensive treatment with neuraminidase resulted in the appearance of a major band with the isoelectric point of 5.6. The purified LTP was rapidly inactivated upon incubation at 37 degrees C due to the denaturation at the "air"-water interface. Various factors promoting or preventing this interfacial denaturation were elucidated. When purified LTP was stored at 4 degrees C, plasma neuraminidase co-purified with LTP became activated, resulting in the gradual desialylation of LTP. It seemed that the LTP preparations of apparent homogeneity are associated with a trace amount of an inactive form of plasma neuraminidase. The inclusion of 4 mM 2-mercaptoethanol or 0.2% EDTA in the storage media completely prevented the activation of plasma neuraminidase. These agents, however, did not significantly inhibit the already activated neuraminidase. When LTP was stored at -20 degrees C in very low ionic strength media, such as 0.001% EDTA (pH 7.4) and at high protein concentrations, the loss of the activity was minimal even after prolonged storage.
Kato
H
H
Department of Food Science, University of Illinois, Urbana 61801.
Nakanishi
T
T
Arai
H
H
Nishida
H I
HI
Nishida
T
T
D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002845:Chromatography; D006801:Humans; D007526:Isoelectric Point; D008970:Molecular Weight; D009439:Neuraminidase / Q000097:blood / Q000378:metabolism; D011489:Protein Denaturation; D013997:Time Factors
D002352:Carrier Proteins; C030363:lipid transfer protein; D009439:Neuraminidase