Human alpha-fetoprotein and albumin: differences in zinc binding

The binding of zinc to both human alpha-fetoprotein (AFP) and albumin isolated from cord serum was studied by Sephadex G-50 gel-filtration chromatography. We found that the total number of binding sites for zinc on AFP and albumin were approximately 16 and 12, respectively. Both graphical analysis and the computer program 'LIGAND' indicate that there are at least two major classes of binding sites for both proteins. Both methods of analysis suggested that there are four to five high-affinity sites for zinc on AFP and only two to three similar sites on albumin. The affinity of zinc for AFP (dissociation constant, Kd, 6-8 X 10(-6) mol/l) was higher than for albumin (Kd, 1-3 X 10(-5) mol/l) for the high-affinity sites. The estimates for the zinc low-affinity binding sites were more uncertain, and several classes of low-affinity binding sites of different affinities might be present in both proteins. The results of our inhibition studies suggest that calcium, copper and lead might also bind with AFP at the zinc-binding sites.     

Technological method of isolation of human alpha-fetoprotein. Estimation of free sulfhydryl and amino groups status in the purified preparation

A new preparative method for the isolation of human alpha-fetoprotein (AFP) from cord blood serum was described. This involves the precipitation of high-molecular compounds with polyethylene glycol and the affinity, ion exchange, and gel permeation chromatographies. Up to several tens of milligrams of the homogeneous AFP can be rapidly (48 h) prepared per isolation cycle in a high yield. The native structure of the isolated AFP was proved by immunochemical analysis. No free sulfhydryl groups were found in the purified AFP, and its reduction with dithiothreitol at 4 degrees C led to the formation of two sulfhydryl groups probably belonging to the Cys18 and Cys67 residues in domain I of the protein molecule. Up to ten amino groups in the purified AFP were shown to be accessible for a mild modification by 3-(2-pyridyldithio)propionic acid N-succinimide ester.     

Human tissue-type plasminogen activator is related to albumin and alpha-fetoprotein

Using a computer program designed to detect evolutionary relationships between proteins, I find that residues 72-110 of the mature sequence of human tissue-type plasminogen activator (t-PA) and 39 residues at the carboxy terminus of human albumin have a comparison score that is 8.8 standard deviation units higher than that obtained with a comparison of randomized sequences of these proteins. The probability (p) of getting this score by chance is approximately 10(-18), indicating that part of t-PA and albumin are derived from a common ancestor. I also find that alpha-fetoprotein, a relative of albumin is related to t-PA. Part of this region on t-PA has been previously shown to be related to epidermal growth factor. t-PA, albumin, alpha-fetoprotein, and epidermal growth factor have diverse biological activities. The finding that these proteins are related suggests some new approaches for studying their functions.     

Generation of free radicals by alloxan in the presence of bovine serum albumin: a role of protein sulfhydryl groups in alloxan cytotoxicity

The interaction of alloxan with bovine serum albumin was studied. When alloxan was incubated with bovine serum albumin, oxygen consumption, H2O2 formation, and diminution of sulfhydryl groups of the protein were observed. During the reaction of alloxan with the protein, superoxide radicals were generated; and under anaerobic conditions, ESR signal of alloxan free radicals was observed. These results strongly suggest that alloxan mediates electron transfer from the protein sulfhydryl groups to oxygen.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet membranes treated with $\text{N-}\text{ethyl}\text{[1?}{}^{14}\text{C}\text{]}\text{maleimide}$, phenyl[²⁰³Hg]mercuric acetate and $\text{p-}\text{chloro}\text{[}{}^{203}\text{Hg}\text{]}\text{mercuribenzoate}$ showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from > 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with ¹⁴C-labeled 5,5′-dithio-bis(2-nitrobenzoic acid) resulted in changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon 5,5′-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

S-mercuric-N-dansyl-cysteine labels the free sulfhydryl groups of human serum cholinesterase

Human serum cholinesterase (BChE) has a putative sulfhydryl group (Cys-66) which is unreactive toward conventional alkylating agents such as iodoacetic acid, raising the possibility that this group is blocked in native BChE. In order to test this further, we examined the reactivity of BChE towards the sulfhydryl-specific reagent S- mercuric-N-dansylcysteine (SMNDC). Stoichiometric binding of 4 mol SMNDC/mol of tetrameric enzyme was observed in fluorescence titration experiments, with retention of catalytic activity. SMNDC remained bound to the protein during SDS-polyacrylamide gel electrophoresis in the absence of reducing agent and the fluorescence pattern observed under U.V. light coincided with the Coomassie Blue stained bands. Addition of excess dithiothreitol to the SMNDC-labeled enzyme resulted in the complete removal of bound SMNDC. Thus, Cys-66 appears to be present in the free sulfhydryl form in BChE, analogous to the corresponding free thiol group (Cys-231) of Torpedo californica acetylcholinesterase. As is the case with the latter species, BChE (labeled or unlabeled) is inactivated by 1.0 x 10(-4)M ZnSO4.     

Distribution of free sulfhydryl and disulfide groups among platelet membrane proteins.

Reactive sulfhydryl and disulfide groups were identified in platelet membrane proteins resolved by sodium dodecyl sulfate-polycrylamide gel electrophoresis. Platelet membranes treated with N-ethyl(1-14C)maleimide, phenyl(203Hg)mercuric acetate and p-chloro(203Hg)mercuribenzoate showed similar patterns of distribution of sulfhydryl groups among the sodium dodecyl sulfate-solubilized membrane proteins. Four major and two minor polypeptides ranging in molecular weight from greater than 200 000 to 20 000 were found to have reactive SH groups. Reduction of membrane proteins by sulfite coupled with subsequent mercaptide formation of the resultant monothiols led to the identification of four polypeptides with disulfide bonds. Reaction of platelet membranes with 14C-labeled 5,5'-dithio-bis(2-nitrobenzoic acid) resulted changes in the distribution profile of the solubilized membrane proteins suggestive of a polymerization process dependent upon, 5,5'-dithio-bis(2-nitrobenzoic acid)-induced intermolecular disulfide interchange.     

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).     

Analysis of free sulfhydryl groups and disulfide bonds in Na+,K+-ATPase

The content of free SH groups and disulfide bonds in the purified pig kidney Na+,K+-ATPase was determined by ammetric titration with silver nitrate. In the native enzyme, most of the free SH groups are masked due to their location in the polypeptide chain regions poorly accessible to SH reagents. Denaturation with 5% SDS and 8 M urea makes these regions accessible thus revealing 22 free SH groups/mol of the protein. After complete blocking of free SH groups with silver ions, 8 SH groups/mol of the protein are being released upon sulfitolysis which indicates the presence of four disulfide bonds in the enzyme. At least one disulfide bridge is located in the alpha-subunit whereas the beta-subunit contains three disulfide bonds.     

The nature of “masked” and “free” sulfhydryl groups in proteins

The relative amounts of readily titrable (‘free’) SH groups, versus that of SH groups titrable readily only following denaturation (‘masked’) has been shown by Szent-Gyorgyi to vary significantly from normal to cancerous organ tissue. It is important therefore to inquire into the causes of the two forms of protein-borne SH. Of the four suggested mechanisms for the “masking” of protein SH groups, (1) sequestration in hydrophobic regions-whether between chain folds or between agglomerated subunits, (2) local steric hindrance, (3) cyclic hydrogen-bonding to local peptide amino acid residues, or (4) covalent bonding as in thiazolines; the first mechanism, that of sequestration in hydrophobic regions appears from present evidence to be the most likely cause. Various spectroscopic, reaction rate, and entropy arguments are presented and compared leading to this conclusion.     

Flow cytometric analysis of rodent epididymal spermatozoal chromatin condensation and loss of free sulfhydryl groups

Flow cytometric measurements were made on acridine orange (AO) and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methyl-coumarin (CPM)-stained epididymal- and vas deferens-derived spermatozoal nuclei to follow the course of chromatin condensation and oxidation of free sulfhydryl groups, respectively, during passage through mouse and rat posttesticular reproductive tracts. Alterations of mouse and rat spermatozoal chromatin during transition from a testicular elongated spermatids to epididymal caput spermatozoa resulted in a threefold loss of DNA stainability with AO. Passage of spermatozoa from the caput to corpus epididymis was accompanied by an approximate 15% loss of DNA stainability, which was maintained at that level throughout passage into the vas deferens. AO stainability of epididymal spermatozoal nuclei was generally independent of -SH group stainability. CPM stainability of rat spermatozoal nuclei free -SH groups was 83%, 18%, and 11% of caput spermatozoal values for corpus, cauda epididymis, and vas deferens, respectively. Comparable values for mice were 69%, 20%, and 18%. CPM stainability was relatively homogeneous for these mouse and rat reproductive tract regions, except mouse corpus epididymis spermatozoal nuclei stained very heterogeneously. Rat spermatozoa detained by ligature up to 7 days in the caput, corpus, and cauda epididymi had CPM staining values equal to or below those of normal vas spermatozoa, indicating that disulfide (S-S) bonding is intrinsic to the spermatozoa and is independent of the epididymal environment. These data suggest that chromatin condensation and loss of spermatozoal DNA stainability during passage from the testis to the vas deferens are independent of S-S bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spin labeling of protein sulfhydryl groups by spin trapping a sulfur radical: application to bovine serum albumin and myosin

Reaction of sulfhydryl-containing compounds, RSH, with Ce4+ in the presence of the spin trap phenyl-N-t-butylnitrone results in the appearance of a nitroxide ESR spectrum, which is greatly diminished if the sulfhydryl group is blocked prior to reaction. The spectra have short lifetimes which can be increased two- to fivefold to half-lives of 5-60 min by prior flushing of the solutions with nitrogen. For small molecules, such as cysteine, N-acetylcysteine, glutathione, and 2-mercaptoethanol, the spectrum is that of a freely rotating nitroxide while for the proteins, bovine serum albumin and myosin, the spectrum is characteristic of a strongly immobilized nitroxide spin label rigidly attached to the protein. Since Ce4+ is reported to oxidize the sulfhydryl group via the thiyl radical, RS, the following reactions are proposed to account for the formation of the nitroxide: (formula; see text) These reactions permit the spin labeling of sulfhydryl proteins such that the nitroxide is much closer to the point of attachment than when using conventional spin-labeling methods.     

The alpha-fetoprotein enhancer region activates the albumin and alpha-fetoprotein promoters during liver development

The four members of the albumin gene family encode the serum transport proteins albumin, α-fetoprotein, α-albumin, and vitamin D-binding protein. These genes are transcribed primarily in the liver with each having a different pattern of developmental expression. The tight linkage of these genes, particularly that of albumin, α-fetoprotein and α-albumin, and their liver-specific expression, has led to the suggestion that these genes share common regulatory elements. To directly examine whether the α-fetoprotein enhancer region could regulate the albumin gene family, expression of these genes was monitored in mice in which this region was deleted by homologous recombination. Our data indicate that this enhancer region is required for α-fetoprotein and albumin activation early in liver development and α-fetoprotein reactivation during liver regeneration, but that albumin, α-albumin, and vitamin D-binding protein expression later in hepatic development is not affected by the absence of these enhancers. We also demonstrate that RNA polymerase II loading on the α-fetoprotein and albumin promoters is reduced in the absence of this enhancer region, indicating a direct role for these enhancers in the assembly of the RNA Polymerase II complex during liver development.