- Email: [email protected]
Protocol for quantitative analysis of paired helical filament solubilization: a method applicable to insoluble amyloids and inclusion bodies
Brain Research Protocols 1 Ž1997. 247–252
Protocol
Protocol for quantitative analysis of paired helical filament solubilization: a method applicable to insoluble amyloids and inclusion bodies Mark A. Smith, Ramanakoppa H. Nagaraj, George Perry
)
Institute of Pathology, DiÕision of Neuropathology, Case Western ReserÕe UniÕersity, 2085 Adelbert Road, CleÕeland, OH 44106-2622, USA Accepted 8 May 1996
Abstract Biochemical studies of amyloidoses have been plagued by the sparing solubility of most amyloids in denaturant solvents. Consequently often only a subclass of amyloid protein is analyzed, a fact that is omitted in most studies. This means that there is often no evaluation of the chemical basis for amyloid insolubility, a factor that may provide valuable information concerning amyloid pathogenesis. We have devised a protocol to quantitatively evaluate the solubilization of insoluble amyloid proteins. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitatiÕe assays to establish solvents that dissolve all protein. Here we describe the application of this protocol to quantitatively establish complete solubilization of the paired helical filaments ŽPHFs. from Alzheimer disease. PHFs are distinct from the other amyloid that defines Alzheimer disease ŽAD., i.e., extracellular amyloid-b deposits of senile plaques, nonetheless, PHFs share all the properties of, and are defined as, an amyloid, i.e., binding Congo red; b-pleated sheet conformation and, most significantly, sparing solubility. PHFs of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with AD. Despite intense efforts to understand the molecular composition of this amyloid, quantitative biochemical analyses have been severely hampered by the extreme insolubility of PHF w8x and by difficulties obtaining a homogeneous PHF fraction. Therefore, to date, all of the published studies on the biochemical composition of insoluble PHFs ŽSDS-insoluble. are qualitative and have provided little or no quantitative data on the proportion of material assayed. Using the solubilization protocol described herein, we found that only high pH was effective in solubilizing PHF while a variety of denaturants and chaotropes resulted in only partial release of component protein. Significantly, the approach is analytical because it allows direct assessment of the significance of two posttranslational modifications in mediating PHF insolubility, i.e., phosphorylation and glycation. Further this protocol provides solubilized protein that can be readily characterized. For example, coupling the method to immunoblotting, ELISA, microsequencing or other analytical techniques would identify components as well as provide a quantitative measure. q 1997 Elsevier Science B.V. Themes: Disorders of the nervous system Topics: Degenerative disease: Alzheimer’sKeywords: Alzheimer disease; Amyloid; Glycation; Paired helical filament; Solubilization
1. Type of research
3. Material
Ø Amyloidoses. Ø Inclusion bodies. Ø Protein chemistry.
3.1. Special equipment
2. Time required (estimate time required) One week for total experimental protocol including amyloid isolationrfractionation, solubilization protocol and quantitation of each extract. ) Corresponding author. Fax: q1 Ž216. 368-8964 or q1 Ž216. 8441810; E-mail: [email protected]
1385-299Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 5 - 2 9 9 X Ž 9 6 . 0 0 0 3 8 - 4
Ø Beckman J-21C and L8-50 centrifuges. Ø LKB laser gel scan ŽPharmacia-LKB.. Ø Bausch and Lomb magnifier Ž=7, calibrated to 0.1 mm.. Ø Kimble Products, Kimax 51w capillary tubes Ž1 mm i.d... 3.2. Chemicals and reagents Ø The types of chemicals and reagents necessary for solubilization will vary from protocol to protocol de-
248
M.A. Smith et al.r Brain Research Protocols 1 (1997) 247–252
pendent upon the nature of the insoluble protein or amyloid. The approach that we have taken is to use a hierarchical extraction technique using successive reagents that are specific for different categories of macromolecular association. In the case of PHF we used extraction agents divided into five categories: Ži. detergent treatment, i.e., 1% SDS; Žii. disulfide bond cleavage, i.e., 1% SDSr1% b-mercaptoethanol Ž bME.; Žiii. ionic disruption, i.e., 5 M NaCl or 0.1 M EDTA; Živ. denaturants, i.e., 8 M urea, 6 M guanidineHCl or 70% formic acid; Žv. alkali treatment, i.e., 0.2 or 1 M NaOH. Ø All reagents from Sigma Chemical Co., St. Louis, MO Žacid phosphatase; acrylamide; alkaline phosphatase; ammonium persulfate; b-mercaptoethanol; Coomassie brilliant blue R250; formic acid; glycine; guanidine; HCl; hydrofluoric acid; NaCl; NaOH; pronase; proteinase K; sarcosyl; sodium lauryl sulfate ŽSDS.; sucrose; TEMED; Tris; urea..
4. Detailed procedure 4.1. (A) Amyloidr protein isolation 4.1.1. Insoluble PHF and control fractions Insoluble PHFs were isolated according to a standard protocol w5x. Briefly, hippocampus and temporal cortex from individual cases, with meninges and white matter removed, was homogenized in 5 vols. of 50 mM Tris-HCl, pH 7.6 containing 0.1 g SDSrg wet tissue. The homogenate was centrifuged at 10 000 = g for 4 h at 228C, the supernatant removed, and the pellet rehomogenized in 1% SDSr10% sucroser50 mM Tris-HCl, pH 7.6 Žbuffer A.. After centrifugation at 25 000 = g for 1 h, the pellet was again homogenized in 1% SDSrbuffer A and layered over a gradient of 12.5 ml each of 1.0 M, 1.2 M and 2.0 M sucroserbuffer A. Following centrifugation for 1 h at 72 000 = g the insoluble PHF fraction at the 1.2–2.0 M interface was collected and used in further experiments. PHF fractions were prepared from Alzheimer disease and identical control fractions were prepared from non-demented young and age-matched controls. A minimum of three cases were individually used for each portion of the study.
4.2. (B) Extraction procedure Extraction with each solvent was performed for 10 min at room temperature with the exception of the alkali treatments which were at 378C or 908C. After each extraction, the sample was centrifuged in a microfuge at 16 000 = g for 30 min at 48C and the supernatant analyzed by 10% polyacrylamide gel electrophoresis ŽSDS-PAGE. according to standard procedures and the pellet, still in the same tube, was then re-extracted in the same agent. All extractions were performed in the same tube to exclude loss of sample on the tube surface. Alkali extractions were neutralized with 1 M Tris-HCl following centrifugation. In initial experiments no significant differences were noted if samples were centrifuged in an airfuge at 170 000 = g for 1 h. 4.3. (C) Quantitation of solubilized protein Extracted proteins were defined as solubilized if present in the supernatant following centrifugation and were able to enter the running gel of SDS-PAGE. Protein was quantitated with an LKB Laser Gel Scan ŽPharmacia-LKB. by densitometric scanning of SDS-PAGE stained with either Coomassie brilliant blue R250 or silver w7x. The entire running gel, but not the stacking gel containing insoluble material unable to enter the gel, was used for densitometric measurements. Densitometric readings were expressed as absorbance units multiplied by area Žmm2 . and data shown are intentionally uncorrected for baseline artifacts produced by each solvent. Chemical treatment often resulted in absorbance readings with no addition of protein. To resolve this problem we sequentially performed each extraction at least 5 times Žin preliminary experiments up to 10 extractions were used for some agents.. The rationale for our approach is that artifactual absorbances produced by the solvent remain constant for all extracts whereas readings of extracted proteins are greatest in the initial extracts. For this reason our data are represented without correcting for baseline artifacts. In practice, we found that essentially all possible extractable protein was removed by the third treatment for the time and agents used. 4.4. (D) Quantitation of insoluble fraction Õolume
4.1.2. Soluble PHF Following the method of Lee et al. w6x, the grey matter of hippocampus and temporal cortex was homogenized in 10 vols. of buffer B Ž0.8 M NaClr10% sucroser50 mM Tris-HCl, pH 7.0. and centrifuged at 27 000 = g for 20 min at 48C. The pellet was re-extracted in buffer B and recentrifuged as previously. The supernatants from both centrifugations were combined, made to 1% Sarcosyl, incubated at 228C for 30 min, centrifuged at 100 000 = g for 1 h at 158C and the pellet used in further experiments.
In parallel, it is essential to evaluate the amyloid resistant to extraction, we quantitated the reduction in fraction volume. After each extraction the PHFs were drawn into capillary tubes Ž1 mm i.d.. and centrifuged at 2500 = g for 30 min. Quantitification of ‘‘ volume’’ was made by measuring the height of the pellet with the reticule of a magnifier ŽBausch and Lomb, =7, calibrated to 0.1 mm. and comparing it to the height of the pellet obtained from the original PHF fraction.
M.A. Smith et al.r Brain Research Protocols 1 (1997) 247–252
4.5. (E) Posttranslational modifications The techniques used in this particular aspect of the protocol must be tailored to those modifications thought or suspected to mediate the insolubility of amyloid protein. For example, in the case of PHF there is significant evidence of two major posttranslational modifications, i.e., phosphorylation and glycation-related bonds. To investigate whether phosphorylation is responsible for mediating insolubility one can specifically dephosphorylate with either alkaline phosphatase, acid phosphatase or hydrofluoric acid w10x. The degree of dephosphorylation can be rapidly assessed by measures of inorganic and organic phosphate groups.
249
An alternate approach is to posttranslationally modify a soluble form of the amyloid so that it generates insoluble aggregates with solubility profiles similar to isolated amyloid. For example, in our study of PHF we found that the in vitro glycation of PHF-t with ribose generates protein aggregates that, like SDS-insoluble PHF, are insoluble in detergent but are soluble at high pH w10x.
5. Results Two complementary measures were used to objectively and quantitatively assess the amount of protein solubilized:
Fig. 1. Schematic representation of supernatant ŽA. and pellet ŽB. analyses used to determine PHF solubility.
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quantity of protein solubilized and reduction of fraction volume. These two approaches are chosen because they are complementary, reproducible, accurate, and not dependent on pure protein fractions. This latter aspect is especially attractive since insoluble amyloid fractions are often difficult to isolate to homogeneity. Our results with PHF show that most protein was solubilized in the first two or three extractions and that subsequent extracts contain baseline values produced by artifacts of the solvent w10x. Extraction in 1% SDS, the buffer employed in preparing the insoluble PHF fraction, confirmed that all elements soluble in SDS were removed by the isolation procedure which contains SDS but that addition of the reducing agent b-mercaptoethanol Ž b-ME. releases a small increment of protein. No further proteins are extracted by the denaturants urea, guanidine or formic acid. In contrast, extraction with alkali extracted large amounts of protein. The validity of this approach was confirmed by amino acid analysis of the solubilized material which showed that over 76% of the protein is solubilized by NaOH at 378C. Analysis of the residual material confirmed these findings. While SDSrb-ME treatment of PHF did not greatly reduce the volume of material Žrelative volume 96% " 1.9., incubation with alkali at 378C or 908C reduced the relative volume of insoluble material to 5.0% " 0.2 and 0.27% " 0.28, respectively ŽFig. 2.. With less than 1% of the fraction remaining, the bulk of PHFs are solubilized by alkali. Alkali could cause base-hydrolysis affecting the densitometric analysis or immunoblot identification of solubilized protein. However, treatment of purified neurofilament and t with alkali as described for the solubilization protocol followed by densitometric scanning showed that the quantity of protein is not significantly reduced and that discrete protein bands at the original molecular weight are still readily apparent w10x. Proteolysis with pronase, proteinase K and to a lesser extent HCl Ž6 M HCl, 958C, 12 h. greatly reduces fraction volume to a similar extent as treatment with alkali, but differ from alkali treatment since no proteins are identifiable by SDS-PAGE analysis. These findings suggest that alkali treatment is not solely solubilizing PHF due to peptide bond hydrolysis but may be affecting cleavage of another protein-based linkage. One possibility is alkali might dephosphorylate PHF. However, we found that the level of organic phosphate is essentially unchanged by the alkali treatments and complete dephosphorylation of PHF with hydrofluoric acid does not reduce the fraction volume or change subsequent solubility of the insoluble PHF in any of the solvents used in this study w10x. Together these data suggest that organic phosphate moieties present in PHF proteins are not responsible for maintaining insolubility. Advanced glycation end products ŽAGE., recently identified in PHF, confer insolubility to a variety of proteins
Žreviewed in w9x.. To determine if AGE could modify soluble PHF to mimic the solubility properties of insoluble PHF we chemically glycated soluble PHF protein by incubation with reducing sugars and showed that soluble PHFs incubated without sugar are readily soluble in SDSrb-ME, whereas soluble PHFs incubated with sugars are relatively insoluble in SDSrb-ME, but are soluble in alkali w10x. Therefore glycation alone can account for the differential solubility between insoluble PHFs and soluble PHFs and in vivo glycation could play an important role in the formation of insoluble PHFs.
6. Discussion 6.1. Interpretation, alternate and supporting protocols Since the protein release assay is dependent on SDSPAGE, analysis of agents that hydrolyze the sample, such as proteolysis or acid hydrolysis while resulting in solubility, would, by our definition, not release protein. Therefore, in such a case, amino acid analysis of the supernatant is appropriate. The results of extraction can also be followed by ultrastructural examination of the fraction. The most unbiased examination can be performed by embedding the fraction in epoxy resin and sectioned in the plane of centrifugation. In this study we quantitatively measure PHF solubilization by coupling a volume-based assay of the residual fraction together with densitometric measures of solubilized protein on SDS-PAGE. The power of this method is the highly reproducible use of quantitative assays as attested by its correspondence to standard assays such as amino acid analysis. We found that PHFs are insoluble in denaturants even those reported by non-quantitative methods to solubilize PHF ŽFig. 1.. The complete solubilization of PHF requires alkali treatment or prolonged proteolysis and this agrees with previous studies reporting the effects of alkali treatment on the structure of PHF ŽFig. 3.. While the PHFs of neurofibrillary tangles have the previously described properties, a separate, though probably related, soluble PHFs ŽA68, PHF-t or SDS-soluble PHF. were biochemically isolated w4x. The presence of two types of PHF leads to the proposition that abnormal posttranslational modifications of t is the first step in PHF formation and that soluble PHFs are the progenitors of insoluble PHFs Žreviewed in w2x.. Further it allows the assessment of the possible role of various posttranslation modifications in PHF insolubility. The suggestion that phosphorylation and glycation are important for PHF insolubility Žreviewed in w1,9x. could be critically evaluated by this technique. The findings here support the latter notion, suggesting that the protein components of the neurofibrillary tangle proteins are composed of cross-linked proteins that are resistant to limited proteolysis since in vitro glycation of soluble PHF transforms it
M.A. Smith et al.r Brain Research Protocols 1 (1997) 247–252
Fig. 2. Densitometric analysis of 5 sequential, left to right, extracts of an insoluble PHF fraction analyzed by SDS-PAGE followed by silver staining. Most protein is solubilized in the first 2–3 extractions. Each panel shows a different category of extracting agent, i.e., it had previously been extracted with the reagent preceding it in the hierarchy Ži–vi; see Section 4.. In Žirii. b-ME, or even more effectively, SDS-b-ME solubilized proteins. Following SDS-b-ME extraction of the PHF; high salt, divalent chelation Žiii. or denaturants Živ. were ineffective in solubilizing proteins. In contrast, NaOH treatment at 378C and even again at 908C was effective in extracting protein Žvrvi.. Extracted protein is expressed as histogram heights in units of absorbance multiplied by area Žmm2 . Ž ns 5; "S.E.M...
into ‘‘insoluble PHF’’ with analogous solubility properties to insoluble PHF isolated from neurofibrillary tangles w10x. The technique described here of quantitatively analyzing the volume of insoluble material as well as the amount of released material is broadly applicable to a number of biological and neurological materials where insolubility confounds their analysis, e.g., amyloidoses and inclusion bodies. Moreover, the use of hierarchical solvents and extraction agents might, as we have found in the case of PHF, shed light onto the biochemical basis by which insolubilization occurs ŽFig. 4.. This protocol can be coupled to immunoblotting w10x and ELISA for immunochemical or to microsequencing for biochemical identification of components. The extent of protein release or volume loss can be correlated with identification of various components. For insoluble PHF we found that many of the identified components were released by SDSrb-ME with only a 4% reduction in volume. We interpreted the slight reduction in volume as removal of associated elements rather than the PHF subunits. In contrast, the quantitative solubilization by alkali was associate with release of t consistent with t being the PHF subunit.
251
Fig. 3. Residual volume-based assay for insoluble PHFs showing that only a very small fraction of material remains following alkali treatment.
6.2. Troubleshooting This protocol provides a highly reproducible measure of protein solubilization, provided the following conditions are observed. First, the residual volume analysis must be performed after restoring the fraction to a defined buffer condition, in this case 1% SDS in 50 M Tris HCl, pH 7.6. Second, if the fraction is isolated by using sucrose gradients, it is necessary to rinse and treat with amylase to remove sucrose prior to beginning the protocol, insoluble
Fig. 4. Volume analysis of soluble PHF protein incubated with ribose for 3 days at 378C. Protein was extracted for 30 min with buffer Žuntreated., SDSr b-ME or 0.2 M NaOH at 378C. Protein is completely solubilized by 0.2 M NaOH at 908C, not shown.
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PHFs contain 6–9 mg sucrosermg protein. Third, it is important that the buffer used for volume analysis contains a surface active agent, e.g., non-ionic or ionic detergent, to minimize adherence to the capillary tube.
7. Quick procedure Ø (A) Amyloidr protein isolation. SDS-insoluble PHF and PHF-t are isolated as previously described w5,6x. Ø (B) Extraction procedure. Replicates of amyloid fractions are sequentially extracted 5 times in the same tube with each solvent. Solvents are hierarchically ranged from detergents, reducing agents, ionic distruptors, denaturants to alkali treatment. After thorough homogenization and centrifugation the supernatant is removed and analyzed. Ø (C) Quantitation of solubilized protein. Extracted proteins are assessed by densitometric scanning of electrophoresed protein stained with Coomasie blue or silver. Ø (D) Quantitation of insoluble fraction Õolume. In parallel, the insoluble material remaining after each extraction can be quantitated by measuring the volume of residual material after centrifugation in a capillary tube. Ø (E) Posttranslational modifications. The effect of removal or addition of specific posttranslational modifications can be assessed using this protocol. For example, protein insolubility mediated through disulfide bonds or phosphorylation could be examined by reducing agents and agents that specifically dephosphorylate protein. Conversely, in vitro modification of native proteins can be used to assess the effects of specific modifications on protein solubility.
8. Essential literature references References: w3,5,7,8,10x.
Acknowledgements This work was supported by National Institutes of Health Grants AG09287 and AG08992, the American Health Assistance Foundation and the American Federation for Aging Research.
References w1x Goedert, M., Sisodia, S.S. and Price, D.L., Neurofibrillary tangles and beta-amyloid deposits in Alzheimer’s disease, Curr. Opin. Neurobiol., 1 Ž1991. 441–447. w2x Goedert, M., Tau protein and the neurofibrillary pathology of Alzheimer’s disease, Trends Neurosci., 16 Ž1994. 460–465. w3x Gonzalez, ´ P.J., Correas, I. and Avila, J., Solubilization and fractionation of paired helical filaments, Neuroscience, 50 Ž1992. 491–499. w4x Greenberg, S.G. and Davies, P., A preparation of Alzheimer paired helical filaments that displays distinct t proteins by polyacrylamide gel electrophoresis, Proc. Natl. Acad. Sci. USA, 87 Ž1990. 5827– 5831. w5x Iqbal, K., Zaidi, T., Thompson, C.H., Merz, P.A. and Wisniewski, H.M., Alzheimer paired helical filaments: bulk isolation, solubility, and protein composition, Acta Neuropathol. (Berl.), 62 Ž1984. 167–177. w6x Lee, V.M., Balin, B.J., Otvos, Jr., L. and Trojanowski, J.Q., A68: a major subunit of paired helical filaments and derivatized forms of normal tau, Science, 251 Ž1991. 675–678. w7x Morrissey, J.H., Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity, Anal. Biochem., 117 Ž1981. 307–310. w8x Selkoe, D.J., Ihara, Y. and Salazar, F.J., Alzheimer’s disease: insolubility of partially purified paired helical filaments in sodium dodycyl sulfate and urea, Science, 215 Ž1982. 1243–1245. w9x Smith, M.A., Sayre, L.M., Monnier, V.M. and Perry, G., Radical AGEing in Alzheimer disease, Trends Neurosci., 18 Ž1995. 172–176. w10x Smith, M.A., Siedlak, S.L., Richey, P.L., Nagaraj, R.H., Elhammer, A. and Perry, G., Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer’s disease, Brain Res., 717 Ž1996. 99–108.
Protocol
Protocol for quantitative analysis of paired helical filament solubilization: a method applicable to insoluble amyloids and inclusion bodies Mark A. Smith, Ramanakoppa H. Nagaraj, George Perry
)
Institute of Pathology, DiÕision of Neuropathology, Case Western ReserÕe UniÕersity, 2085 Adelbert Road, CleÕeland, OH 44106-2622, USA Accepted 8 May 1996
Abstract Biochemical studies of amyloidoses have been plagued by the sparing solubility of most amyloids in denaturant solvents. Consequently often only a subclass of amyloid protein is analyzed, a fact that is omitted in most studies. This means that there is often no evaluation of the chemical basis for amyloid insolubility, a factor that may provide valuable information concerning amyloid pathogenesis. We have devised a protocol to quantitatively evaluate the solubilization of insoluble amyloid proteins. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitatiÕe assays to establish solvents that dissolve all protein. Here we describe the application of this protocol to quantitatively establish complete solubilization of the paired helical filaments ŽPHFs. from Alzheimer disease. PHFs are distinct from the other amyloid that defines Alzheimer disease ŽAD., i.e., extracellular amyloid-b deposits of senile plaques, nonetheless, PHFs share all the properties of, and are defined as, an amyloid, i.e., binding Congo red; b-pleated sheet conformation and, most significantly, sparing solubility. PHFs of neurofibrillary tangles are the most striking intraneuronal change seen within the brains of patients with AD. Despite intense efforts to understand the molecular composition of this amyloid, quantitative biochemical analyses have been severely hampered by the extreme insolubility of PHF w8x and by difficulties obtaining a homogeneous PHF fraction. Therefore, to date, all of the published studies on the biochemical composition of insoluble PHFs ŽSDS-insoluble. are qualitative and have provided little or no quantitative data on the proportion of material assayed. Using the solubilization protocol described herein, we found that only high pH was effective in solubilizing PHF while a variety of denaturants and chaotropes resulted in only partial release of component protein. Significantly, the approach is analytical because it allows direct assessment of the significance of two posttranslational modifications in mediating PHF insolubility, i.e., phosphorylation and glycation. Further this protocol provides solubilized protein that can be readily characterized. For example, coupling the method to immunoblotting, ELISA, microsequencing or other analytical techniques would identify components as well as provide a quantitative measure. q 1997 Elsevier Science B.V. Themes: Disorders of the nervous system Topics: Degenerative disease: Alzheimer’sKeywords: Alzheimer disease; Amyloid; Glycation; Paired helical filament; Solubilization
1. Type of research
3. Material
Ø Amyloidoses. Ø Inclusion bodies. Ø Protein chemistry.
3.1. Special equipment
2. Time required (estimate time required) One week for total experimental protocol including amyloid isolationrfractionation, solubilization protocol and quantitation of each extract. ) Corresponding author. Fax: q1 Ž216. 368-8964 or q1 Ž216. 8441810; E-mail: [email protected]
1385-299Xr97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 1 3 8 5 - 2 9 9 X Ž 9 6 . 0 0 0 3 8 - 4
Ø Beckman J-21C and L8-50 centrifuges. Ø LKB laser gel scan ŽPharmacia-LKB.. Ø Bausch and Lomb magnifier Ž=7, calibrated to 0.1 mm.. Ø Kimble Products, Kimax 51w capillary tubes Ž1 mm i.d... 3.2. Chemicals and reagents Ø The types of chemicals and reagents necessary for solubilization will vary from protocol to protocol de-
248
M.A. Smith et al.r Brain Research Protocols 1 (1997) 247–252
pendent upon the nature of the insoluble protein or amyloid. The approach that we have taken is to use a hierarchical extraction technique using successive reagents that are specific for different categories of macromolecular association. In the case of PHF we used extraction agents divided into five categories: Ži. detergent treatment, i.e., 1% SDS; Žii. disulfide bond cleavage, i.e., 1% SDSr1% b-mercaptoethanol Ž bME.; Žiii. ionic disruption, i.e., 5 M NaCl or 0.1 M EDTA; Živ. denaturants, i.e., 8 M urea, 6 M guanidineHCl or 70% formic acid; Žv. alkali treatment, i.e., 0.2 or 1 M NaOH. Ø All reagents from Sigma Chemical Co., St. Louis, MO Žacid phosphatase; acrylamide; alkaline phosphatase; ammonium persulfate; b-mercaptoethanol; Coomassie brilliant blue R250; formic acid; glycine; guanidine; HCl; hydrofluoric acid; NaCl; NaOH; pronase; proteinase K; sarcosyl; sodium lauryl sulfate ŽSDS.; sucrose; TEMED; Tris; urea..
4. Detailed procedure 4.1. (A) Amyloidr protein isolation 4.1.1. Insoluble PHF and control fractions Insoluble PHFs were isolated according to a standard protocol w5x. Briefly, hippocampus and temporal cortex from individual cases, with meninges and white matter removed, was homogenized in 5 vols. of 50 mM Tris-HCl, pH 7.6 containing 0.1 g SDSrg wet tissue. The homogenate was centrifuged at 10 000 = g for 4 h at 228C, the supernatant removed, and the pellet rehomogenized in 1% SDSr10% sucroser50 mM Tris-HCl, pH 7.6 Žbuffer A.. After centrifugation at 25 000 = g for 1 h, the pellet was again homogenized in 1% SDSrbuffer A and layered over a gradient of 12.5 ml each of 1.0 M, 1.2 M and 2.0 M sucroserbuffer A. Following centrifugation for 1 h at 72 000 = g the insoluble PHF fraction at the 1.2–2.0 M interface was collected and used in further experiments. PHF fractions were prepared from Alzheimer disease and identical control fractions were prepared from non-demented young and age-matched controls. A minimum of three cases were individually used for each portion of the study.
4.2. (B) Extraction procedure Extraction with each solvent was performed for 10 min at room temperature with the exception of the alkali treatments which were at 378C or 908C. After each extraction, the sample was centrifuged in a microfuge at 16 000 = g for 30 min at 48C and the supernatant analyzed by 10% polyacrylamide gel electrophoresis ŽSDS-PAGE. according to standard procedures and the pellet, still in the same tube, was then re-extracted in the same agent. All extractions were performed in the same tube to exclude loss of sample on the tube surface. Alkali extractions were neutralized with 1 M Tris-HCl following centrifugation. In initial experiments no significant differences were noted if samples were centrifuged in an airfuge at 170 000 = g for 1 h. 4.3. (C) Quantitation of solubilized protein Extracted proteins were defined as solubilized if present in the supernatant following centrifugation and were able to enter the running gel of SDS-PAGE. Protein was quantitated with an LKB Laser Gel Scan ŽPharmacia-LKB. by densitometric scanning of SDS-PAGE stained with either Coomassie brilliant blue R250 or silver w7x. The entire running gel, but not the stacking gel containing insoluble material unable to enter the gel, was used for densitometric measurements. Densitometric readings were expressed as absorbance units multiplied by area Žmm2 . and data shown are intentionally uncorrected for baseline artifacts produced by each solvent. Chemical treatment often resulted in absorbance readings with no addition of protein. To resolve this problem we sequentially performed each extraction at least 5 times Žin preliminary experiments up to 10 extractions were used for some agents.. The rationale for our approach is that artifactual absorbances produced by the solvent remain constant for all extracts whereas readings of extracted proteins are greatest in the initial extracts. For this reason our data are represented without correcting for baseline artifacts. In practice, we found that essentially all possible extractable protein was removed by the third treatment for the time and agents used. 4.4. (D) Quantitation of insoluble fraction Õolume
4.1.2. Soluble PHF Following the method of Lee et al. w6x, the grey matter of hippocampus and temporal cortex was homogenized in 10 vols. of buffer B Ž0.8 M NaClr10% sucroser50 mM Tris-HCl, pH 7.0. and centrifuged at 27 000 = g for 20 min at 48C. The pellet was re-extracted in buffer B and recentrifuged as previously. The supernatants from both centrifugations were combined, made to 1% Sarcosyl, incubated at 228C for 30 min, centrifuged at 100 000 = g for 1 h at 158C and the pellet used in further experiments.
In parallel, it is essential to evaluate the amyloid resistant to extraction, we quantitated the reduction in fraction volume. After each extraction the PHFs were drawn into capillary tubes Ž1 mm i.d.. and centrifuged at 2500 = g for 30 min. Quantitification of ‘‘ volume’’ was made by measuring the height of the pellet with the reticule of a magnifier ŽBausch and Lomb, =7, calibrated to 0.1 mm. and comparing it to the height of the pellet obtained from the original PHF fraction.
M.A. Smith et al.r Brain Research Protocols 1 (1997) 247–252
4.5. (E) Posttranslational modifications The techniques used in this particular aspect of the protocol must be tailored to those modifications thought or suspected to mediate the insolubility of amyloid protein. For example, in the case of PHF there is significant evidence of two major posttranslational modifications, i.e., phosphorylation and glycation-related bonds. To investigate whether phosphorylation is responsible for mediating insolubility one can specifically dephosphorylate with either alkaline phosphatase, acid phosphatase or hydrofluoric acid w10x. The degree of dephosphorylation can be rapidly assessed by measures of inorganic and organic phosphate groups.
249
An alternate approach is to posttranslationally modify a soluble form of the amyloid so that it generates insoluble aggregates with solubility profiles similar to isolated amyloid. For example, in our study of PHF we found that the in vitro glycation of PHF-t with ribose generates protein aggregates that, like SDS-insoluble PHF, are insoluble in detergent but are soluble at high pH w10x.
5. Results Two complementary measures were used to objectively and quantitatively assess the amount of protein solubilized:
Fig. 1. Schematic representation of supernatant ŽA. and pellet ŽB. analyses used to determine PHF solubility.
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quantity of protein solubilized and reduction of fraction volume. These two approaches are chosen because they are complementary, reproducible, accurate, and not dependent on pure protein fractions. This latter aspect is especially attractive since insoluble amyloid fractions are often difficult to isolate to homogeneity. Our results with PHF show that most protein was solubilized in the first two or three extractions and that subsequent extracts contain baseline values produced by artifacts of the solvent w10x. Extraction in 1% SDS, the buffer employed in preparing the insoluble PHF fraction, confirmed that all elements soluble in SDS were removed by the isolation procedure which contains SDS but that addition of the reducing agent b-mercaptoethanol Ž b-ME. releases a small increment of protein. No further proteins are extracted by the denaturants urea, guanidine or formic acid. In contrast, extraction with alkali extracted large amounts of protein. The validity of this approach was confirmed by amino acid analysis of the solubilized material which showed that over 76% of the protein is solubilized by NaOH at 378C. Analysis of the residual material confirmed these findings. While SDSrb-ME treatment of PHF did not greatly reduce the volume of material Žrelative volume 96% " 1.9., incubation with alkali at 378C or 908C reduced the relative volume of insoluble material to 5.0% " 0.2 and 0.27% " 0.28, respectively ŽFig. 2.. With less than 1% of the fraction remaining, the bulk of PHFs are solubilized by alkali. Alkali could cause base-hydrolysis affecting the densitometric analysis or immunoblot identification of solubilized protein. However, treatment of purified neurofilament and t with alkali as described for the solubilization protocol followed by densitometric scanning showed that the quantity of protein is not significantly reduced and that discrete protein bands at the original molecular weight are still readily apparent w10x. Proteolysis with pronase, proteinase K and to a lesser extent HCl Ž6 M HCl, 958C, 12 h. greatly reduces fraction volume to a similar extent as treatment with alkali, but differ from alkali treatment since no proteins are identifiable by SDS-PAGE analysis. These findings suggest that alkali treatment is not solely solubilizing PHF due to peptide bond hydrolysis but may be affecting cleavage of another protein-based linkage. One possibility is alkali might dephosphorylate PHF. However, we found that the level of organic phosphate is essentially unchanged by the alkali treatments and complete dephosphorylation of PHF with hydrofluoric acid does not reduce the fraction volume or change subsequent solubility of the insoluble PHF in any of the solvents used in this study w10x. Together these data suggest that organic phosphate moieties present in PHF proteins are not responsible for maintaining insolubility. Advanced glycation end products ŽAGE., recently identified in PHF, confer insolubility to a variety of proteins
Žreviewed in w9x.. To determine if AGE could modify soluble PHF to mimic the solubility properties of insoluble PHF we chemically glycated soluble PHF protein by incubation with reducing sugars and showed that soluble PHFs incubated without sugar are readily soluble in SDSrb-ME, whereas soluble PHFs incubated with sugars are relatively insoluble in SDSrb-ME, but are soluble in alkali w10x. Therefore glycation alone can account for the differential solubility between insoluble PHFs and soluble PHFs and in vivo glycation could play an important role in the formation of insoluble PHFs.
6. Discussion 6.1. Interpretation, alternate and supporting protocols Since the protein release assay is dependent on SDSPAGE, analysis of agents that hydrolyze the sample, such as proteolysis or acid hydrolysis while resulting in solubility, would, by our definition, not release protein. Therefore, in such a case, amino acid analysis of the supernatant is appropriate. The results of extraction can also be followed by ultrastructural examination of the fraction. The most unbiased examination can be performed by embedding the fraction in epoxy resin and sectioned in the plane of centrifugation. In this study we quantitatively measure PHF solubilization by coupling a volume-based assay of the residual fraction together with densitometric measures of solubilized protein on SDS-PAGE. The power of this method is the highly reproducible use of quantitative assays as attested by its correspondence to standard assays such as amino acid analysis. We found that PHFs are insoluble in denaturants even those reported by non-quantitative methods to solubilize PHF ŽFig. 1.. The complete solubilization of PHF requires alkali treatment or prolonged proteolysis and this agrees with previous studies reporting the effects of alkali treatment on the structure of PHF ŽFig. 3.. While the PHFs of neurofibrillary tangles have the previously described properties, a separate, though probably related, soluble PHFs ŽA68, PHF-t or SDS-soluble PHF. were biochemically isolated w4x. The presence of two types of PHF leads to the proposition that abnormal posttranslational modifications of t is the first step in PHF formation and that soluble PHFs are the progenitors of insoluble PHFs Žreviewed in w2x.. Further it allows the assessment of the possible role of various posttranslation modifications in PHF insolubility. The suggestion that phosphorylation and glycation are important for PHF insolubility Žreviewed in w1,9x. could be critically evaluated by this technique. The findings here support the latter notion, suggesting that the protein components of the neurofibrillary tangle proteins are composed of cross-linked proteins that are resistant to limited proteolysis since in vitro glycation of soluble PHF transforms it
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Fig. 2. Densitometric analysis of 5 sequential, left to right, extracts of an insoluble PHF fraction analyzed by SDS-PAGE followed by silver staining. Most protein is solubilized in the first 2–3 extractions. Each panel shows a different category of extracting agent, i.e., it had previously been extracted with the reagent preceding it in the hierarchy Ži–vi; see Section 4.. In Žirii. b-ME, or even more effectively, SDS-b-ME solubilized proteins. Following SDS-b-ME extraction of the PHF; high salt, divalent chelation Žiii. or denaturants Živ. were ineffective in solubilizing proteins. In contrast, NaOH treatment at 378C and even again at 908C was effective in extracting protein Žvrvi.. Extracted protein is expressed as histogram heights in units of absorbance multiplied by area Žmm2 . Ž ns 5; "S.E.M...
into ‘‘insoluble PHF’’ with analogous solubility properties to insoluble PHF isolated from neurofibrillary tangles w10x. The technique described here of quantitatively analyzing the volume of insoluble material as well as the amount of released material is broadly applicable to a number of biological and neurological materials where insolubility confounds their analysis, e.g., amyloidoses and inclusion bodies. Moreover, the use of hierarchical solvents and extraction agents might, as we have found in the case of PHF, shed light onto the biochemical basis by which insolubilization occurs ŽFig. 4.. This protocol can be coupled to immunoblotting w10x and ELISA for immunochemical or to microsequencing for biochemical identification of components. The extent of protein release or volume loss can be correlated with identification of various components. For insoluble PHF we found that many of the identified components were released by SDSrb-ME with only a 4% reduction in volume. We interpreted the slight reduction in volume as removal of associated elements rather than the PHF subunits. In contrast, the quantitative solubilization by alkali was associate with release of t consistent with t being the PHF subunit.
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Fig. 3. Residual volume-based assay for insoluble PHFs showing that only a very small fraction of material remains following alkali treatment.
6.2. Troubleshooting This protocol provides a highly reproducible measure of protein solubilization, provided the following conditions are observed. First, the residual volume analysis must be performed after restoring the fraction to a defined buffer condition, in this case 1% SDS in 50 M Tris HCl, pH 7.6. Second, if the fraction is isolated by using sucrose gradients, it is necessary to rinse and treat with amylase to remove sucrose prior to beginning the protocol, insoluble
Fig. 4. Volume analysis of soluble PHF protein incubated with ribose for 3 days at 378C. Protein was extracted for 30 min with buffer Žuntreated., SDSr b-ME or 0.2 M NaOH at 378C. Protein is completely solubilized by 0.2 M NaOH at 908C, not shown.
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PHFs contain 6–9 mg sucrosermg protein. Third, it is important that the buffer used for volume analysis contains a surface active agent, e.g., non-ionic or ionic detergent, to minimize adherence to the capillary tube.
7. Quick procedure Ø (A) Amyloidr protein isolation. SDS-insoluble PHF and PHF-t are isolated as previously described w5,6x. Ø (B) Extraction procedure. Replicates of amyloid fractions are sequentially extracted 5 times in the same tube with each solvent. Solvents are hierarchically ranged from detergents, reducing agents, ionic distruptors, denaturants to alkali treatment. After thorough homogenization and centrifugation the supernatant is removed and analyzed. Ø (C) Quantitation of solubilized protein. Extracted proteins are assessed by densitometric scanning of electrophoresed protein stained with Coomasie blue or silver. Ø (D) Quantitation of insoluble fraction Õolume. In parallel, the insoluble material remaining after each extraction can be quantitated by measuring the volume of residual material after centrifugation in a capillary tube. Ø (E) Posttranslational modifications. The effect of removal or addition of specific posttranslational modifications can be assessed using this protocol. For example, protein insolubility mediated through disulfide bonds or phosphorylation could be examined by reducing agents and agents that specifically dephosphorylate protein. Conversely, in vitro modification of native proteins can be used to assess the effects of specific modifications on protein solubility.
8. Essential literature references References: w3,5,7,8,10x.
Acknowledgements This work was supported by National Institutes of Health Grants AG09287 and AG08992, the American Health Assistance Foundation and the American Federation for Aging Research.
References w1x Goedert, M., Sisodia, S.S. and Price, D.L., Neurofibrillary tangles and beta-amyloid deposits in Alzheimer’s disease, Curr. Opin. Neurobiol., 1 Ž1991. 441–447. w2x Goedert, M., Tau protein and the neurofibrillary pathology of Alzheimer’s disease, Trends Neurosci., 16 Ž1994. 460–465. w3x Gonzalez, ´ P.J., Correas, I. and Avila, J., Solubilization and fractionation of paired helical filaments, Neuroscience, 50 Ž1992. 491–499. w4x Greenberg, S.G. and Davies, P., A preparation of Alzheimer paired helical filaments that displays distinct t proteins by polyacrylamide gel electrophoresis, Proc. Natl. Acad. Sci. USA, 87 Ž1990. 5827– 5831. w5x Iqbal, K., Zaidi, T., Thompson, C.H., Merz, P.A. and Wisniewski, H.M., Alzheimer paired helical filaments: bulk isolation, solubility, and protein composition, Acta Neuropathol. (Berl.), 62 Ž1984. 167–177. w6x Lee, V.M., Balin, B.J., Otvos, Jr., L. and Trojanowski, J.Q., A68: a major subunit of paired helical filaments and derivatized forms of normal tau, Science, 251 Ž1991. 675–678. w7x Morrissey, J.H., Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity, Anal. Biochem., 117 Ž1981. 307–310. w8x Selkoe, D.J., Ihara, Y. and Salazar, F.J., Alzheimer’s disease: insolubility of partially purified paired helical filaments in sodium dodycyl sulfate and urea, Science, 215 Ž1982. 1243–1245. w9x Smith, M.A., Sayre, L.M., Monnier, V.M. and Perry, G., Radical AGEing in Alzheimer disease, Trends Neurosci., 18 Ž1995. 172–176. w10x Smith, M.A., Siedlak, S.L., Richey, P.L., Nagaraj, R.H., Elhammer, A. and Perry, G., Quantitative solubilization and analysis of insoluble paired helical filaments from Alzheimer’s disease, Brain Res., 717 Ž1996. 99–108.