Studies on factor VIII-related protein. II. Estimation of molecular size differences between factor VIII oligomers.

Human factor VIII-related protein was isolated from cryoprecipitate by agarose (Sepharose CL-2B) gel filtration. Electrophoresis on SDS-2% polyacrylamide-0.5% agarose gels revealed size heterogeneity of factor VIII-related protein which was similar to that shown by SDS-1% agarose gel electrophoresis and electron microscopy. The apparent molecular weights were compared with those of crosslinked IgM oligomers and corresponded to values of up to 20 . 10(6) for factor VIII eluting close to the void volume of our gel filtration column. Measurement of mobility intervals on electrophoretic gels suggested a constant size difference between adjacent bands. Smaller aggregates were found in later eluates from Sepharose columns as well as following partial reduction of factor VIII with cysteine. In order to compare the size difference between small and large aggregates of factor VIII-related protein we calibrated the SDS-2% polyacrylamide-0.5% agarose gels with factor VIII which had been crosslinked with dimethyl suberimidate and subsequently disulfied-reduced with 2-metcaptoethanol. By combination of calibration ranges, constant intervals were measured for large and smaller factor VIII aggregates. The interval between any neighboring protein bands, which were immunologically identified as factor VIII-related protein, was equal to the dimer of the basic factor VIII subunit chain. We conclude that factor VIII aggregates correspond to multimers of a dimeric molecule, i.e. pairs of the basic subunit chain.     

Purification of rabbit and human serum paraoxonase.

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.     

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.     

Zein of maize grain: II — the charge heterogeneity of free subunits

The subunits present as monomers in unreduced zein and isolated as fraction M by gel filtration, were chromatographed on sulfoethyl-cellulose. Three major subfractions were detected and characterized. Each of them, submitted to electrophoresis at pH 3.5, migrated as a single band corresponding to each of the three major electrophoretic forms seen in fraction M at the same pH. The presence of lysine in some polypeptides, suggested by amino acid composition data, was confirmed by electrophoretic analysis of carbamylated subfractions at pH 4.5. At pH 8.9 each subfractions was further resolved into three cationic bands in starch gel and three (or more) anionic bands in polyacrylamide gel. The same fractionation was also obtained by submitting the major electroforms of fraction M, as isolated at pH 3.5, to isoelectric focusing. Based on these observations, the most probable distributions of basic amino acids in subunits detected by electrophoresis at pH 8.9 were specified and compared to those recently published for several zein clones. The presence per polypeptide chain of three carboxyl groups and occasionally of one lysine would be a feature of zein originating from maize hybrid Inra 260.     

Studies on factor VIII-related protein. I. Ultrastructural and electrophoretic heterogeneity of human factor VIII-related protein.

Human factor VIII-related protein precipitates with specific heterologous anti-bodies directed against purified factor VIII and supports ristocetin-induced aggregation of washed platelets. We purified human factor VIII from cryoprecipitate by subsequent gel filtration on crosslinked large-pore agarose. Factor VIII-related protein appeared as a large aggregate following electrophoresis on 3% polyacrylamide gels in the presence of sodium dodecyl sulfate (SDS). The same material was separated into multiple bands (molecular weight in excess of several millions) following electrophoresis on SDS-1% agarose gels. After complete disulfide reduction of factor VIII-related protein and electrophoresis on SDS-5% polyacrylamide gels a single subunit chain (Mr approximately equal to 200 000) was revealed. Analysis of this protein, in its non-reduced state, by negative contrast electron microscopy showed filaments of markedly variable size. The calculated molecular weight of such filaments ranged from about 0.6.10(6) to 20.10(6). We conclude that size heterogeneity is an essential feature of human factor VIII-related protein.     

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.     

Analysis of rat serum apolipoproteins by isoelectric focusing. I. Studies on the middle molecular weight subunits.

Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.     

Use of the hydrophobic probe Nile red for the fluorescent staining of protein bands in sodium dodecyl sulfate-polyacrylamide gels.

In a previous work (J.-R. Daban, M. Samsó, and S. Bartolomé, Anal. Biochem. 199, 162-168, 1991) we observed that, in the presence of the detergent sodium dodecyl sulfate (SDS), diverse types of proteins produced a high increase in the fluorescence intensity of the hydrophobic probe 9-diethylamino-5H-benzo[alpha]-phenoxazine-5-one (Nile red). This enhancement of Nile red fluorescence was observed at SDS concentrations lower than the critical micelle concentration (CMC) of this detergent in the buffer (0.025 M Tris and 0.192 M glycine, pH 8.3) currently used in SDS-polyacrylamide gel electrophoresis. This observation led us to introduce a modification in the typical (U. K. Laemmli, Nature 227, 680-685, 1970) SDS-polyacrylamide gels, in which the SDS concentration in the gel after electrophoresis is lower than the CMC of this detergent but high enough to maintain the stability of the protein-SDS complexes in the bands. The staining of these modified gels with Nile red produces very high fluorescence in the protein-SDS bands and low background fluorescence. The Nile red staining method described in this paper is very rapid (i.e., the bands can be visualized and photographed within 6 min after the electrophoretic separation) and has a high sensitivity, similar to that obtained with the covalent fluorophores rhodamine B isothiocyanate and carboxytetramethyl-rhodamine succinimidyl ester also investigated in this work. Furthermore, our quantitative estimates indicate that most of the protein bands stained with Nile red show similar values of the fluorescence intensity per unit mass.     

Alterations of mouse embryo cells during in vitro aging

Trichocysts from the ciliated protozoan Paramecium aurelia have been solubilized in aqueous solution at neutral pH by heating to temperatures of 70 °C for 10 min. The product of such solubilization appears identical to that achieved by the previous method using trichocysts solubilized in sodium dodecyl sulfate (SDS) when examined by SDS polyacrylamide disc gel electrophoresis. While the solubilization of the trichocyst is virtually total in distilled water, no more than 20% of the total protein solubilizes in the presence of salts even in amounts as low as 0.05 M. Heat solubilized trichocysts show two principal components when run on standard 10% polyacrylamide disc gels and two bands when run on SDS polyacrylamide disc gels. The relationship between the two bands seen on standard gels and the two bands seen on SDS gels is unknown at this time. These results represent the first successful solubilization of this organelle in aqueous solution at neutral pH as opposed to previous reports requiring strong denaturants such as SDS, guanidine hydrochloride, and pH extremes. While the solubilization of trichocysts by heat most probably results in denaturation of the constituent proteins, it does occur in aqueous solution without the use of strong denaturants or pH extremes allowing the use of standard analytical procedures not possible in the presence of these reagents.     

Biochemical isolation and physiological identification of the egg- laying hormone in Aplysia californica

It has been determined that the bag cells of Aplysia californica produce two polypeptide species that comigrate on electrophoretic gels containing sodium dodecyl sulfate. By this separation procedure both species can be assigned a molecular weight of approximately 6,000. One of these molecules has an Rf of 0.65 on alkaline discontinuous electrophoresis gels, an isoelectric point at pH 4.8, a gel filtration molecular weight of approximately 12,000, and has no known biological function. The other does not enter alkaline disk gels, has an isoelectric point at approximately pH 9.3, shows a gel filtration molecular weight consistent with that determined by SDS gel electrophoresis, and is the egg-laying hormone.     

A facile method for the isolation and preparation of proteins and peptides for sequence analysis in the picomolar range

We report a new and facile extraction method of proteins and polypeptides in the range of 100 to 1 kDa previously separated by high-resolution SDS/polyacrylamide-gel electrophoresis. Proteins and polypeptides obtained by chemical or proteolytic cleavage of proteins can directly be applied to high-sensitivity N-terminal amino-acid sequence analysis by gas-phase sequencing. The Coomassie Blue-stained protein bands are eluted from the gel slices with 0.1 M sodium acetate buffer, pH 8.5, 0.1% SDS in high yield and directly applied to the filter disc of the gas-phase sequencer. The superior efficiency for the isolation of proteins and polypeptides from polyacrylamide gels for microsequencing has been documented by a quantitative comparison of the procedure described here and the favoured electroblot-transfer method using 14C-labeled marker proteins. This highly efficient isolation has been successfully reproduced and applied to the analysis of a variety of proteins and peptides with rather divergent physical properties, particularly to hydrophobic peptides isolated from SDS/polyacrylamide gels. The electrophoretic transfer onto activated glass filters. Immobilon membranes (polyvinylidene-difluoride membranes), siliconized or chemically activated glass fiber supports can be omitted. The method considerably simplifies and speeds up the isolation, and improves the sensitivity as compared to the electroblotting procedures due to the reproducibly high recoveries.     

Electrophoretic properties of sodium dodecyl sulfate and related changes in its concentration in SDS-polyacrylamide gel electrophoresis.

Sodium dodecyl sulfate(SDS) in a protein sample solution migrates in SDS-polyacrylamide gel electrophoresis as a band with a mobility higher than those of protein bands. Behind this band, which is mostly composed of SDS micelles, SDS concentration is raised uniformly in a gel column as a result of the retardation effect of the gel matrix on SDS micelles. Electrophoretic patterns of SDS were obtained when SDS was omitted from various portions of the gel electrophoretic system.     

Purification of RNAase II by preparative polyacrylamide gel electrophoresis.

Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease is activated by K+ and Mg2+ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913--922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacrylamide gels gives a single band. A molecular weight of 76 000 +/- 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 +/- 2000, 33 000 +/- 2000, and 26 000 +/- 1000. The enzyme thus appears to consist of three dissimilar subunits.     

Electron microscopy of human factor VIII/Von Willebrand glycoprotein: effect of reducing reagents on structure and function

The structure of native and progressively reduced human factor VIII/von Willebrand factor (FVIII/vWF) was examined by electron microscopy and SDS gel electrophoresis and then correlated with its biological activities. Highly resolved electron micrographs of well-spaced, rotary- shadowed FVIII/vWF molecules showed their structure to consist of a very flexible filament that contains irregularly spaced small nodules. Filaments ranged from 50 to 1,150 nm with a mean length of 478 nm and lacked fixed, large globular domains as seen in fibrinogen and IgM. A population of multimeric FVIII/vWF species ranging in molecular weight from 1 to 5 million daltons and differing in size alternately by one and two subunits was observed on SDS-2% polyacrylamide-0.5% agarose gel electrophoresis. With progressive reduction of disulfide bonds by dithiothreitol (DTT), the electron microscopic size of FVIII/vWF decreased in parallel with increased electrophoretic mobility on SDS- agarose gels; between 0.1 and 0.5 mM DTT its structure changed from predominantly fibrillar species to large nodular forms. A 50% loss of vWF specific activity and FVIII procoagulant activity occurred at 0.4 mM DTT and 1 mM DTT, respectively, corresponding to the reduction of 4 and 12 disulfide bonds of the 62 disulfides per 200,000-dalton subunit. We conclude that reduction of a few critical disulfide bonds results in a major structural change by electron microscopy and a concomitant loss of approximately 50% of the vWF function.     

DNA-dependent RNA polymerases from Acanthamoeba castellanii. Comparative subunit structures of the homogeneous enzymes.

The constituent polypeptides of the three classes of DNA-dependent RNA polymerase from Acanthamoeba castellanii were compared by several electrophoretic methods. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) reveals that a number of polypeptide components of the isozymes have identical molecular weights. Two-dimensional electrophoresis (isoelectric focusing in 8 M urea:SDS-polyacrylamide gel electrophoresis) demonstrates that the polypeptides of identical molecular weights also have identical isoelectric pH values. These polypeptides were also coincident after electrophoresis in 8 M urea at acidic or basic pH values followed by a second electrophoretic separation in the presence of SDS. By these criteria, subunits of molecular weight 13,300, 15,500, 17,500, 22,500, 37,000, and 39,000 are indistinguishable in polymerase I and III. The 13,300, 15,500, and 22,500 subunits are also shared by the class II polymerase. In addition, electrophoresis in 8 M urea under basic conditions reveals microheterogeneity in the 17,500 molecular weight subunit. The strikingly similar pattern of common subunits between yeast and Acanthamoeba suggests that a universal arrangement of functional units may be an essential feature of the eukaryotic polymerases.     

Silver staining of high molecular weight proteins on large-pore polyacrylamide gels

A large-pore gel for electrophoresis in the presence of sodium dodecyl sulfate, composed of 2.55% polyacrylamide crosslinked with 2.75% methylenebisacrylamide, is described. This gel has a resolving power for very high molecular weight proteins and can be stained with silver. The gel is suitable for fractionation of factor VIII/von Willebrand factor directly from plasma samples. Visualization by silver staining revealed a series of covalently bound multimers with molecular weights of up to 8 X 10(6). The procedure described should be useful also for studies on other very high molecular weight proteins and nucleic acids.     

Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis

A scheme for electroblotting of individual unstained protein bands from SDS/polyacrylamide gels and subsequent amino acid sequence analysis is described. Principal features are: detection of the polypeptide bands by visualization with KCl; electroblotting of excised gel pieces that correspond to the protein bands only; blotting onto polybrene-pretreated glass-fiber filter discs (12 mm diameter) placed in an electrophoretic concentrator. A high yield over all steps from gel application through electrophoresis, blotting, gas-phase sequencer degradation, and phenylthiohydantoin analysis is obtained with several different types of polypeptide (combined average yield over all steps 20%, spread 10-50%). Background is low and samples can be stored under vacuum for long periods after blotting.     

Specific dimerization of the light chains of human immunoglobulin

1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b).     

Resolving Power: A Quantitative Measure of Electrophoretic Resolution

Resolving power is a quantitative measure of the ability of an electrophoretic system to separate DNA (and other) molecules of similar size. It is a dimensionless quantity, and hence facilitates comparison of the performance of electrophoretic systems that operate very differently. Resolving power can be determined as a function of molecular length from experimental data consisting of a series of completely resolved bands on a gel or blot; closely spaced bands are not required. We discuss factors such as the mass of DNA in a particular band and the spatial resolution of the system used to image the distribution of DNA on a gel or blot that, while not an intrinsic part of the electrophoretic system, may influence the observed resolving power. We derive an empirical global dispersion function that applies both to images of gels obtained after a fixed time of electrophoresis of all the samples and to images obtained as each species reaches a detector located at a fixed distance from the starting well. We use this dispersion function to show that the improvement in resolving power produced by extending the time or distance of electrophoresis in a static, uniform electric field asymptotically approaches a limiting value that is a function of the length of the DNA. When plotted as a function of molecular length, this limiting value defines an envelope that characterizes the intrinsic limits of performance of a particular electrophoretic system (e.g., electric field strength, gel type and concentration, buffer, temperature). Comparing the resolving power of static field agarose gel electrophoresis as routinely practiced for separating DNA molecules from 10³ to 10⁵ bp long with other electrophoretic schemes suggests that significant improvements should be achievable.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.