SECOND EDITION
`
`Protein
`Methods
`
`Daniel M. Bollag / Michael D. Rozycki / Stuart J. Edelstein
`
`1 of 148
`
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`Exhibit 1017
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`

`

`Protein Methods
`
`Second Edition
`
`DANIEL M. BOLLAG
`Merck Research Labo rato ri es
`West Point, Pennsylvani a
`
`MICHAEL D. ROZYCKI
`Depa rtment of C hemistry
`The Henry H. H oyt Laboratory
`Princeto n University
`Princeton. New Jersey
`
`STUART J. EDELSTEIN
`Depa rtment of Biochemistry
`University o f Geneva
`Geneva, Switzerla nd
`
`~WILEY-LISS
`A JOHN WTLEY & SONS, INC., PUBLICATION
`• Chichester • Brisbane • Toronto • Singapore
`New York
`
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`Address all Inquiries to the Publisher
`Wiley-Liss, Inc .. 605 Third Avenue, New York, NY 10158-0012
`
`Copyright © 1996 Wiley-Uss, Inc,
`
`Printed in the Unite d State$ o f America.
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`
`All rights reserved. This book is protected hy coryright. No part ofit. except brief excerpts for
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`from the publisher.
`
`While the authors. editors. ,,n<l p uhlls hn helievc 1ha1 drug selection and dosage and the
`speci fication and usage of equipment and devices. as set forth in this hook are in accord with
`current recommendations and practice at the time of publication. t hey accept no legal re·
`sponsibility fo r any errors or omissions. and make no warranty. expres~ or implied. with
`respect to material containt:d herein. In vie" of ongoing rese-an:h. <'quipment modifications.
`changes in governmental regu lations and the cons1an1 flow of information relating to drug
`the rapy. drug re:ictions. and the use ofe4uipmen1 and dcvk--cs. the reader is urged to review
`and evaluate the information provided in the package insert vr in~tructions for each drug.
`piece of e4uipmenl. or device for. Hmong other th ings. any changes in the in~tructions or
`in<lirnrion of dosage or u~age and ror uddctl warnings and precautions.
`
`Library of Congress Cataloging in Publication Data
`
`Bollag. Dan iel M.
`P rotein methods / D.iniel M. Bo\Jag. Mi.:had D. Rozycki.
`Stuart J. Edelstein. -
`Jnd ed.
`cm.
`p.
`Includes bib liographical refNences and indl.'.X.
`ISBN 0-47 1-1 1837-0 (cloth ; alk. paper)
`I. Proteins-Purifo:ation. 1. Proteins- Research-Methodology.
`I. Edelstein. Stuart J.
`ll. Ro?.)'Cki. Mi.:hnel D. Ill. Title.
`QPS.51.E:!3 1996
`~74.19'145-dc20
`10 9 Ii 7 h 5 4 .l 1
`
`'U,- 1408.,
`
`3 of 148
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`

`Chapter 1
`
`Preparation for Protein Isolation
`
`I. Introduction
`
`11. Buffers
`A. Buffer Characteristics
`B . Preparation of Buffers
`C. Concentration Effects of Buffer on pH
`D. Limitations of Certain Buffers
`E. Preventing Buffer Contamination
`F. Water Purity
`
`III. Salts, Metal Ions, and Chelators
`A. Ionic Strength
`B. Divalent Cations
`C. Chelators
`
`IV. Reducing Agents
`A. General Considerations
`B. Specific Recommendations
`
`V . Detergents
`A. Introduction
`B. Classes of Detergents
`C. Protocol for Membrane Protein Solubilization
`
`VI. Protein Environment
`A. Surface Effects
`B. Temperature
`C. Storage
`
`VII. Protease Inhibitors
`A. Common Inhibitors
`B. A Sample Broad Range Protease Inhibitor Cocktail
`
`VIII. References
`
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`2
`
`Protein Methods
`
`I. Introduction
`
`This book is devoted to laboratory techniques for the analysis and
`separation of proteins. Proteins are an extremely heterogeneous class of
`biological macromolecules. They are often unstable when not in their
`native environment, which in itself varies considerably among cell
`compartments and extracellular fluids. Of the many types of proteins, we
`can distinguish between those that are soluble or membrane-bound, those
`with catalytic or purely structural roles, and those with various post(cid:173)
`translational modifications.
`
`Each protein may have specific requirements once it is extracted from
`its nonnal biological milieu. If these requirements are not satisfied, the
`protein can rapidly lose its ability to carry out specific functions, and an
`already limited lifetime may be drastically reduced. Thus, determination
`of these requirements has often been a major hurdle in protein
`characterization. In some cases, the difficulty has been to stabilize the
`protein against external proteolysis, while in other cases the problem has
`been to maintain ligand-binding or enzymatic activity. Solutions to these
`problems are highly
`individual.
`Nonetheless, some fundamental
`parameters must be considered by anyone studying proteins.
`In this
`chapter, we discuss a number of these parameters and attempt to provide
`general guidelines or sources of infonnation for laboratory work with
`proteins.
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`Preparation for Protein Isolation
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`3
`
`JI. Buffers
`
`A. Buff er Characteristics
`
`• A buffer is defined as a mixture of an acid and its conjugate base
`which can reduce changes in solution pH when acid or alkali are
`added. The selection of an appropriate buffer is important in
`order to maintain a protein at the desired pH and to ensure
`reproducible experimental results. A rudimentary description of
`key concepts behind buffering, such as pH and pKa, can be found
`in tpe Calbiochem "Buffers" booklet and in Stryer (1988, pp. 41-
`42).
`
`• There are eight important characteristics to consider when
`selecting a buffer (adapted from Scopes, 1982):
`1. pKa value (see Table 1.1)
`2. pKa variation with temperature
`3. pKa variation upon dilution
`4. Solubility
`5. Interaction with other components (such as metal ions and
`enzymes)
`6. Expense
`7. UV absorbance
`8. Permeability through biological membranes
`
`• Some General Observations
`
`l. Ideally, different buffers with a similar pKa should be tested
`to determine whether there are undesired
`interactions
`between a certain buffer and the protein under investigation
`(Blanchard, 1984).
`
`2. Once a buffer is chosen, it is best to work at the lowest
`reasonable concentration to avoid nonspecific ionic strength
`effects. A 50 mM buffer is a good starting point.
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`4
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`Protein Methods
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`3. The useful buffering range diminishes significantly beyond
`1 pH umt on either side of the pKa. Note that many
`enzymes are irreversibly denatured at extreme pH values
`(Tipton and Dixon, 1979).
`
`4. The physiological pH in most animal cells is 7.0 - 7.5 at
`37°C. Due to the effect of temperature, this value rises to
`close to 8.0 near 0°C (Scopes, 1982).
`
`5. The buffer of choice also depends on the methods employed;
`• For gel filtration chromatography, almost any buffer can
`be chosen that is compatible with the protein of interest.
`• For anion exchange chromatography. cationic buffers such
`as Tris are preferred.
`• For cation exchange or hydroxyapatite chromatography,
`anionic buffers
`such as phosphate are preferred
`(Blanchard, 1984).
`
`6. 'Good's' buffers (for example, MES, PIPES, MOPS) were
`developed by Good and colleagues ( 1966) to be biologically
`inert, to have low UV absorbance, and to be minimally
`affected by temperature or iomc strength.
`
`7. Buffer mixtures with wide buffering ranges at constant ionic
`strength are described by Ellis and Morrison (1982).
`
`low
`for
`8. A description of buffers and cryosolvents
`temperature conditions is found in Fink and Geeves ( 1979).
`
`9. All chemical products should be reagent grade or higher.
`
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`Preparation for Protein Isolation
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`5
`
`B. Preparation of Buffers
`
`• In principle, the pH of a solution can be adjusted directly at the
`temperature at which the buffer is to be used. However, this
`requires that the pH electrode be standardized at the working
`temperature, often 4°C, while in practice. buffer is usually
`prepared at room temperature and the pH adjusted so that it will
`be correct after the solution is brought to the desired temperature.
`It should be noted that temperature effects on buffer pH may be
`large. A notable example is Tris, which bas a pKa that changes
`from 8.06 at 25°C to 8.85 at 0°C [Blanchard, 1984]). An
`experimental solution should be tested for its pH after all the
`components (e.g. EDTA, DIT, Mg2+-) have been added since the
`pH may change after their addition.
`
`• Unless other instructions are given, assume that the pH of a
`buffer is adjusted down with HCl and up with either NaOH or
`KOH.
`
`• The basicity of tetramethylammonium hydroxide is equivalent to
`NaOH or KOH. Tetramethylammoniurn hydroxide should be
`used in adjusting the pH of a buffer for a reaction which requires
`the complete absence of mono-, di-, or trivalent metal ions
`(Calbiochem "Buffers" bookJet).
`
`• By convention, the molarity of a buffer corresponds to the acid
`component. Thus, 1 M Tris-acetate, pH 4.8 means that I M
`acetic acid is titrated with about 0.5 M Tris base to give the final
`pH. However. in common usage, just the opposite is often
`intended: a solution of Tris base may be titrated with acetic acid.
`Or, even more confusingly, Tris-HCI (the "acid" form) may be
`titrated with acetic acid, resulting jn a three-component system of
`Tris, chloride, and acetate. Be aware of this potential for
`ambiguity when trying to duplicate recipes from the literature.
`
`• If both protonated and unprotonated forms of a buffer are readily
`available, solutions of the two forms at the same concentration
`can be mixed until the desired pH is obtained. It is preferable to
`use established tables or calculations for mixing components,
`although verification with a pH meter is always advisable as in
`the following example:
`
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`6
`
`Protein Methods
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`Preparation of a phosphate buffer between pH 5.8 and 7 .8
`(Calbiochem ''Buffers" book.let):
`
`Stock Solution A (0.2 M NaH2PO4) ~
`Dissolve 27.6 g NaH2PO4 to make I liter in deioruzed
`water.
`
`Stock Solution B (0.2 M NazIIPO4):
`Dissolve 28.4 g NazHPO4 to make. I liter in deionized
`water.
`
`mi
`5.8
`5.9
`6.0
`6.1
`6.2
`6.3
`6.4
`6.5
`6.6
`6.7
`6.8
`
`%A
`92.0
`90.0
`87.7
`85.0
`81.5
`77.5
`73.5
`68.5
`62.5
`56.5
`5LO
`
`%B
`8.0
`10.0
`12.3
`15.0
`19.5
`22.5
`26.5
`31.5
`37.5
`43.5
`49.0
`
`Jill % A
`6.9
`45.0
`7.0 39.0
`7.1
`33.0
`7.2 28.0
`23.0
`7.3
`7.4
`1.9.0
`7.5
`16.0
`7.6
`13.0
`7.7
`10.5
`7.8
`8.5
`
`%B
`55.0
`61.0
`67.0
`72.0
`77.0
`81.0
`84.0
`87.0
`89.5
`91.5
`
`Note: pH values are approximate and will vary according
`to exact temperature and the accuracy at which components
`are mixed and measured. Therefore, they should be
`verified with a pH meter. However, pH electrodes
`themselves are subject to significant variability. Thus, for
`maximum reproducibility, it is best to adjust pH by adding
`known amounts of each component, rather than titrating
`each time wjtb a pH meter.
`
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`Preparation for Protein Isolation
`
`7
`
`Table 1.1
`PKa Values of Common Biological Buffers
`
`Buffer Name
`
`2-(N-Morpholino)ethanesulfonic acid
`Dimethylarsinic acid
`
`3-(N-Morpholino )propanesulfonic acid
`
`pKfl_"
`2.15
`3.06
`3.75
`4.21
`4.76
`4.76
`5.23
`5.40
`5.64
`6.15
`6.27
`6.35
`[B is-(2-hydroxyethy l)imino ]tris(hydroxymethyl )methane 6.46
`6.59
`N-2-Acetamidoiminodiacetic acid
`Piperazine-N,N'-bis(2-ethanesulfonic acid)
`6.76
`1,3-Bis[tris(hydroxymethyl)methylantino ]propane
`6.80
`N-2-Acetamido-2-aminoethanesulfonic acid
`6.90
`6.95
`7.20
`7.20
`2-[Tris(hydroxymethyl)methylamino ]ethanesulfonic acid 7 .50
`N-2-Hydroxyethylpiperazine-N'-2-ethanesuJfonic acid
`7.55
`N-2-Hydroxyethylpiperazine-N'-3-propane-sulfonic acid 8.00
`Tris(hydroxymethyl)aminomethane
`8.06
`N-[Tris(hydroxymethyl)methyl]glycine
`8. I 5
`8.25
`8.35
`
`Trivial Naine
`Phosphate (pKa1)
`Citrate (pKa1)
`Formate
`Succinate (pKat)
`Cilrate (pKa2)
`Acetate
`Pyridine
`Citrate (pKa3)
`Succinate (Pl<a2)
`MES
`Cacodylate
`Carbonate (pKai)
`BIS-Tris
`ADA
`PIPES
`BIS-Tris propane
`ACES
`Imidazole
`MOPS
`Phosphate (pKa2)
`TES
`HEPES
`HEPPS (EPPS)
`Tris
`Tricine
`Glycylglycine
`Bicine
`TAPS
`
`N,N-Bis(2-hydroxyethyl)glycine
`3- (fTris(hydroxymethyl)methyIJamino} propane-suJ fonic
`acid
`
`8.40
`9.23
`Borate
`9.25
`Ammonia
`9.55
`CHES
`Glycine
`9.78
`I 0.33
`Carbonate (PKa2)
`10.40
`CAPS
`I 2.43
`Phosphate (pKa3)
`• Values from Calbiochem "Buffers" booklet or B lanchard ( 1984).
`
`Cyclohexylaminoethanesulfonic acid
`
`.3-(cyclohexylamino)propanesulfonic acid
`
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`

`8
`
`Protein Methods
`
`C. Concentration Effects of Buffer on pH
`
`• It is useful to prepare buffers as lOx or 1 OOx stocks. This permits
`smaller storage volumes and the addition of bactericidal agents
`such as 0.02% sodium azide which are diluted to insignificant
`levels before use (Scopes, 1982). Saturating solubilities of some
`buffers at 0°C (for full chemical names, see Table 1.1):
`
`MES
`PIPES
`MOPS
`TES
`HEPES
`Tris
`Phosphate
`
`0.65 M
`2.3 M
`3.0M
`2.6M
`2.3 M
`2.4M
`2.5 M (as K+ salt)
`
`• Note that dilution of concentrated stock buffer solution may
`change the pH. For example, a buffer with 0.1 M NaH2P04 and
`0.1 M N~HP04 is pH 6.7. Tenfold dilution raises the pH to 6.9
`while after one hundredfold dilution it is 7 .0 (Tipton and Dixon,
`1979).
`
`• The pH of Tris decreases by 0.1 unit per tenfold dilution
`(Calbiochem "Buffers" booklet).
`
`D. Limitations of Certain Buffers
`
`Buffers are often present at the highest concentration of all
`components in a protein solution and may have significant effects
`on a protein or enzyme. Buffers composed of inorganic compounds
`(phosphate, borate, bicarbonate) may interact with enzymes (or
`their substrates), affecting their activities. Most seriously, some
`buffers form coordination complexes with di- and trivalent metal
`ions resulting in proton release, lower pH, chelation of the metal,
`and formation of insoluble complexes. Buffers with low metal
`binding constants such as PIPES, TES, HEPES, and CAPS are
`preferred
`for
`studying enzymes with metal
`requirements
`(Blanchard, 1984).
`
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`Preparation for Protein Isolation
`
`9
`
`• Phosphate:
`1. is a feeble buffer in the pl-I range 8 - 11 ;
`2. precipitates or binds many polyvalent cations;
`3. inhibits a large variety of.enzymes, including kinases,
`phosphatases, dehydrogenases, and other enzymes with
`phosphate esters as substrates (Blanchard, 1984);
`4. exhibits a dependence of pK on buffer dilution (see Section C).
`
`• Citrate binds to some proteins and forms metal complexes
`(Scopes, 1982).
`
`• Cacodylate is toxic (Scopes, 1982).
`
`• Carbonate has lihlited solubility and, since it is in equilibrium
`with CO2, studies must be carried out in a closed system
`(Blanchard, 1984).
`
`• ADA absorbs light at wavelengths up to 260 nm and binds metal
`ions (Good et al., 1966).
`
`• MOPS interferes with the Lowry protein assay, but not with
`either the Bradford or Bicinchoninic Acid assays (see Chapter 3).
`
`• HEPES:
`1. interferes with the Lowry protein assay, but not with either the
`Bradford or Bicinchoninic Acid assays (see Chapter 3);
`2 . as for all piperazine-based Good buffers (HEPES, EPPS,
`PIPES; see Good et al., 1966) forms radicals under various
`conditions and should be avoided in systems where redox
`processes are being studied (Grady et al., 1988).
`
`• Tris:
`1. is a poor buffer below pH 7 .O;
`2. possesses a potentially reactive primary amine;
`3. participates in various enzymatic reactions such as that
`catalyzed by alkaline phosphatase;
`4. passes through biological membranes (Calbiochem "Buffers"
`booklet);
`5. is affected by buffer concentration and temperature (see
`Section C above).
`
`• Borate forms complexes with mono- and oligosaccharides,
`nucleic acids and pyridine nucleotides, and glycerol (Blanchard,
`1984).
`
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`

`10
`
`Protein Methods
`
`E. Preventing Buffer Contamination
`
`• Phosphate-buffered solutions are highly susceptible to microbial
`contamination. However, 1 M phosphate stock solutions do not
`usually become contaminated with bacteria (Schleif and
`Wensink, 1981).
`
`• Filtering the buffer through a sterile ultrafiltration device may be
`useful for preventing bacterial or fungal growth, especially at pH
`6 - 8 (Blanchard, 1984).
`
`• To prevent buffer contamination during storage, 0.02% (3 mM)
`sodium azide is often used. Sodium azide does not interact
`significantly with proteins at this concentration. Note: Sodium
`azide should be used with caution in the presence of heavy metal
`cations, as it can dry down into highly unstable salt crystals
`(Rozycki and Bartha, 1981).
`
`• Refrigeration helps to reduce buffer contamination.
`
`F. Water Purity
`
`Water is the primary ingredient in almost every laboratory
`solution. Most contaminating substances are removed by
`distillation and deionization. but traces of some compounds
`sometimes remain and reliable measurements with protein
`solutions may be affected. A description of various treatment
`systems for high-level purification of water for laboratory research
`can be found in Oanzi (1984).
`
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`

`Preparation for Protein Isolation
`
`11
`
`m. Salts, Metal Ions, and Chelators
`
`A. Ionic Strength: 0.1 - 0.2 M KCl or NaCl simulates physiological
`conditions for many applications (O'Sullivan and Smithers, 1979),
`
`B. Divalent Cations
`
`If a complex is formed between the buff er and a divalent cation
`such as Ca2+ or Mg2+, the capacity for buffering hydrogen ions is
`reduced.
`In addition,. the availability of the metal ions to
`participate in an enzymatic reaction may be diminished. Thus,
`beware of buffers with affinities for metals.
`
`L. A void Tris buffers when a metal cofactor is required for
`protein activity or stability. In 100 mM Tris with 2 mM
`Mn2+, 29% of the metal is chelated (Morrison, 1979).
`
`2. For purposes of reproducibility, if working with an ATP(cid:173)
`binding enzyme, add Mg2+ in 1 mM excess over ATP to
`ensure that essentially all ATP is present as Mg·ATP (Watts,
`1973).
`
`C. Chelators
`
`When it is necessary to fimit metal effects, specific metal ion
`chelators should be used. Metal ion che]ators also inactivate
`metalloproteases.
`
`1. To eliminate trace amounts of heavy metals in buffers, 0.1 -
`5 mM ethylene diamine tetraacetic acid (EDT A)
`is
`commonly used (Scopes, 1982).
`
`2. The most commonly used chelating agents are EDTA and
`ethylene bis(oxyethylenenitrilo)tetraacetic acid (EGTA).
`While EDT A displays strong and nonspecific affinity for a
`variety of metals, the affinity of EGT A for calcium is
`significantly higher
`than
`its affinity for magnesium,
`permitting the preferential sequestering of calcium
`in
`solutions with EGT A (Blanchard, 1984 ).
`
`3. o-Phenanthroline chelates zinc, while m-phenanthroline does
`not (Todhunter, 1979).
`
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`

`12
`
`Protein Methods
`
`IV. Reducing Agents
`
`A. General Considerations
`
`reducing compounds, notably
`the cell, various
`Within
`glutathione, prevent protein oxidation. Once the cell has been
`disrupted, care must be taken to counteract effects due to increased
`contact with oxygen and dilution of naturally occurring reducing
`agents. Many proteins lose activity when oxidized, although this
`activity may sometimes be restored by reduction of critical thioJ
`groups. The presence of divalent cations may accelerate the
`formation of disulfide bonds (Scopes, 1982).
`
`B. Specific Recommendations
`
`• 2-Mercaptoethanol, which is easy to use since it may be stored as
`a solution at 4 °C, must be used at a concentration of 5 - 20 mM.
`introduction
`into
`the buffer, 2-
`Within 24 hours of its
`mercaptoethanol becomes oxidized, after which it may accelerate
`protein inactivation (Scopes, 1982).
`
`• Dithiothreitol (OTT or Cleland's reagent) is supplied as a powder
`and must be stored at -20°C as a stock solution. DIT may be
`used at 0.5 - 1.0 mM, and oxidation results in the formation of a
`stable intramolecular disulfide which does not endanger protein
`sulfhydryls.
`
`• A good strategy is to use 2-mercaptoethanol at a 1: 1000 dilution
`(about 12 mM) during a protein preparation, but 1 - 5 mM DTI
`for long term storage (Scopes, 1982).
`
`• Note that certain enzymes are sensitive to reduction (Schleif and
`Wensinek, 1981).
`
`15 of 148
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`

`

`Preparation for Protein Isolation 13-
`
`V. Detergents
`
`A. Introduction
`
`Detergents are used most often for the extraction and
`purification of membrane proteins, which otherwise are usually
`jnsoluble in aqueous solution. A number of classes of detergents
`may be used and some general guidelines for the solubilization and
`stabilization of membrane proteins are presented in this section.
`
`Detergents are amphlphilic molecules with substantial
`solubility in water. With the exception of bile salts, the
`hydrophobic portion of the molecule usually consists of a linear or
`branched hydrocarbon "tail" whereas the hydrophilic "head" may
`have one of a number of very different chemical structures. An
`important property of detergents is the formation of micelles,
`which are clusters of detergent molecules in whlch the hydrophilic
`head portions face outward. Solubilized membrane proteins form
`mixed micelles with detergent.
`The hydrophobic
`(or
`transmembrane) domain of the protein is shielded from contact
`with the aqueous buffer by detergent molecules. The critical
`micelle concentration (CMC) is defined as the lowest detergent
`concentration at whlch micelles form. A detergent with a high
`CMC (e.g., octyl glucoside) will return to the monomeric state
`upon dilution below this concentration, thus permitting rapid
`In addition, the total
`removal of the detergent by dialysis.
`molecular weight of the protein plus the miceJle may be important
`for dialysis, gel filtration chromatography, and electrophoresis
`under non-denaturing conditions. Factors affecting the CMC
`include temperature, pH, ionic strength, presence of multivalent
`ions or organic solvents, and detergent purity.
`
`Although the presence of high concentrations of detergent often
`results in protein denaturation, sometimes the subsequent removal
`of detergent allows the protein to renature. If protein denaturation
`is a problem, nonionic detergent concentrations below 0.1 % are not
`usually harmful to proteins (Scopes, 1982).
`
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`

`14
`
`Protein Methods
`
`B. Classes of Detergents
`
`Consult Table 1.2 for specific properties of detergents.
`
`• Ionic Detergents
`
`Ionic detergents contain head groups with either positive
`charges (cationic detergents) or negative charges (anionic
`Ionic detergents have the disadvantage of being
`detergents).
`highly denaturing. However, they pennit the separation of
`proteins into their monomeric forms, facilitating molecular
`weight detennination.
`
`1. Sodium or Lithium Dodecyl Sulfate (SDS, LiDS)
`
`Of these two anionic detergents, lithium dodecyl sulfate
`(LiDS) has the advantage that it is soluble at 4°C while sodium
`dodecyl sulfate (SDS) is not. As much as 10 mg or more of
`LiDS may be necessary for complete solubilization of one mg
`of membrane protein (Boehringer Mannheim Catalog). Note
`that the use of these detergents in the presence of potassium
`buffers or ammonium sulfate may lead to their precipitation at
`room temperature.
`The critical micelle concentration is
`dramatically affected by the salt concentration; for SDS, the
`CMC drops from 8 mM in the absence of salt to 0.5 mM in 0.5
`M NaCl (Helenius et al., l979).
`
`2. Sodium Cholate and Sodium Deoxycholate
`
`Sodium cholate and sodium deoxycholate (DOC) are
`anionic detergents that are less denaturing than other ionic
`detergents (Harlow and Lane, 1988). Two classes of micelles,
`termed primary (containing up to 9 molecules) and secondary
`(9 - 60 molecules), occur
`above
`the critical micelle
`concentration. In contrast to other detergents, free cholate or
`deoxycholate monomers continue
`to accumulate above the
`CMC. The pKa of these detergents is between 8 and 9, and
`precipitation of the detergent in its acid form is a problem
`below pH 7.5. Divalent cations cause DOC precipitation.
`
`17 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`Preparation for Protein Isolation
`
`15
`
`• Nonionic Detergents
`
`Nonionic detergents have uncharged hydrophilic head
`groups. As a result, they are less likely to disrupt protein(cid:173)
`protein interactions and are particularly useful for isolating
`functional protein complexes. Nonionic detergents are far less
`denaturing than ionic detergents~ however, protein aggregation
`may occur in the pres1,;nce of these detergents.
`
`1. Triton X-100 (Polyoxyethylene [9-10] p+octyl phenol)
`
`Many proteins retain their activity in 1 - 3% Triton X-100.
`A tenfold or greater excess of Triton X-100 to membrane lipid
`(w/w) may be required to solubilize membrane proteins. Triton
`X-100 has a strong absorbance at 280 nm.
`
`2. Triton X-114 (Polyoxyethylene [7-8] p-t-octyl phenol)
`
`Triton X-114 (2%) added to a protein solution has the
`property of causing a separation between the detergent and the
`aqueous phases at temperatures above 20°C, its cloud point.
`Hydrophilic proteins relnain in the aqueous phase while
`integral membrane proteins may be recovered in the detergent
`phase (Bordier, 1981).
`
`3. Octyl glucoside ( 1-O-n-octyl-~-D-glucopyranoside)
`
`Octyl glucoside (OG) is better defined chemically than
`Triton and therefor
`ts may be more reproducible with this
`- 45 mM G is often sufficient to solubilize
`detergent.
`membrane protems.
`as a high CMC (see Table 1.2) and is
`more easily removed from solution than is Triton.
`
`4. Tween 20 (PEG [20] sorbitan monolaurate)
`
`. Tw~en 2~ is c~mmonly ~sed to block_ nons1,>ecific protein
`mteract1ons m sobd phase 1mmunochenustry (ELISA, RIA,
`immunoblotting). Tween 20 has a very low CMC.
`
`18 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`16
`
`Protein Methods
`
`• Zwitterionic Detergents
`
`Zwitterionic detergents contain head groups with both
`positive and negative charges. This class of detergents is more
`efficient than nonionic detergents at overcoming protein(cid:173)
`protein interactions while causing less protein denaturation
`than ionic detergents.
`
`I. CHAPS
`(3-[(Cholamidopropyl)dimethyl-ammonio]-1 -
`propanesulfonate)
`
`ion exchange
`interfere with
`CHAPS does not
`chromatography or isoelectric focusing.
`Proteins in
`solutions containing CHAPS may be frozen safely.
`
`2. Zwittergent 3-14
`
`19 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`Table 1.2
`
`Characteristics of Common Detergents
`
`Detergent
`
`Sodium Dodecyl Sulfate
`Lithium Dodecyl Sulfate
`Sodium Cholate
`Sodium Deoxycholate
`Triton X-100
`Triton X-l 14
`Octyl glucoside
`Tween 20
`Tween 80
`Brij 35
`CHAPS
`Zwittergent 3-14
`
`Molecular
`Weight
`288.Sd
`272.4
`43Jf
`433r
`about 628a
`about 543d
`292.4a
`123Qf
`1310f
`12()()f
`614.9d
`364d
`
`Critical MiceUe
`Concentration
`7 - IO mM, 0.23%d
`6-8 mM,02%a
`3 - 10 rnM, 0.2%f
`l - 2 mM, 0.57%f
`0.3 mM, 0.02%c
`0.35 mM, 0.02%
`15 - 25 mM,0.5o/oB
`60 µM, 0.006%f
`10 µM, 0.0013%f
`90 µM, 0.0J%e
`4 - 8 mM, 0.5%f
`0.3 mM, 0.011 %
`
`Concentration for
`Solubilization
`
`> 10 mg/mg proteina
`
`0.2 - 0.6 mg/mg proteinb
`see text (section V.B.)
`-
`-20-45 mM3
`
`6- 10 mMD
`
`Micelle Size
`
`18 kd
`
`1.8 kd
`4.2 kd
`90kd
`
`8.0 kd
`
`76kd
`49 kd
`6.0kd
`30kd
`
`a • Boehringer Mannheim Catalog
`b - Helenius and Simons, 1975
`c - Hjelmeland and Chrambach, 1984
`d - Calbiochem Catalog
`e - Helenius et al., 1979
`f - Harlow and Lane, 1988
`
`20 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`18
`
`Protein Methods
`
`C. Protocol for Membrane Protein Solubilization
`
`• Experimental Steps (from Hjelmeland and Chrambach, 1984 ):
`
`1. Prepare crude membrane fraction at a protein concentration
`of about 10 mg/ml in 50 mM buffer, 0.15 M KCl at 4°C.
`
`2. Prepare detergent stock solution (10%, w/v) in the same
`buffer.
`
`3. Make dilutions of the c.rude membrane fraction (about 5
`mg/ml) in buffer containing the following amounts of
`detergent: 0.01 %, 0.03%, 0.1 %, 0.3%, l %, 3%.
`
`4. Stir gently for 1 hr at 4°C (avoid foaming or sonication).
`
`5. Centrifuge at 100,000 x g for 1 hr at 4°C.
`
`6 . Remove supernatant and resuspend pellet in an equal volume
`of buffer containing the same detergent concentration.
`
`7. Determine the protein concentration of each fraction (see
`Chapter 3).
`
`8. Determine the enzyme activity in each fraction.
`
`21 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`Preparation for Protein Isolation
`
`• General Comments
`
`l. The detergent concentration which yields the highest soluble
`protein content and activity should be used for further
`studies. If unsatisfactory results are obtained with a variety
`of different detergents, mixtures of detergents may be tried.
`Low yields of protein activity may be improved by the
`addition of glycerol (25 - 50%, v/v), reducing agents (1 mM
`DTT or 5 mM j3-mercaptoethanol), chelating agents (1 mM
`EDTA), or protease inhibitors (75 µg/ml PMSF, 20 µg/ml
`leupeptin or pepstatin; see Section VII below).
`
`2. Sometimes, solubilization with phosphate (0.1 - 0.2 M) is
`successful if KCl solubilization does not work.
`
`J. Protein solubili.zation very often occurs near the detergent
`CMC (Hjelmeland and Chrambach, 1984).
`
`4. For separating soluble protein from integral membrane
`proteins, the method of Bordier (1981 ) using the nonionic
`detergent Triton X - 114 may be employed. Extraction and
`separation in Triton X-114 may be used as a first step in the
`purification of a membrane protein (this step rapidly
`separates the membrane proteins from lysosomal proteases).
`Detergent exchange may be performed during ion-exchange
`chromatography.
`
`5. The interactions of membrane-associated proteins with
`membranes may also be disrupted by exposing
`the
`membrane preparation to conditions of high salt (0.15 - 2.0
`M KC!). high or low pH, high doses of chelating agents ( 10
`mM EDTA or EGTA), or denaturing agents such as urea or
`guanidine HCl (6 - 10 M) (van Renswoude and Kempf,
`1984).
`
`6. The definition of a soluble protein is not alway$ clear. The
`ability of a protein to remain in the supernatant after a one
`hour centrifugation at 105,000 x g is affected by both the
`solution density and temperature. Another test that may be
`applied is gel filtration chromatography (Hjelmeland and
`Chrambach, 1984 ).
`
`22 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`20
`
`Protein Methods
`
`VL Protein Environment
`
`A. Surface Effects
`
`Dilute protein solutions often lose activity quickly, possibly via
`denaturation on surfaces such as glassware, but this effect can be
`prevented by inclusion of high levels of another protein, commonly
`bovine serum albumin (BSA).
`Ideally, to avoid introducing a
`"contaminating'' protein, dilute protein solutions should be rapidly
`concentrated. However, since enzyme reactions are sometimes
`assayed with protein concentrations as low as 1 µg/rol which may
`lead to rapid inactivation, addition of BSA may be necessary. lo
`addition, loss of the purified protein due to nonspecific adhesion
`onto glass surf aces (1 µg of protein is absorbed on 5 cm2 of a glass
`surface)
`is significantly diminished when
`the solution
`is
`supplemented with BSA. At least 0.1 mg/ml BSA should be used
`in assay mixtures, while stored protein may contain as much as 10
`mghnl BSA (Scopes, 1982).
`
`B. Temperature
`
`An enzyme's reaction velocity roughly doubles with a
`temperature increase of l0°C (for example between l8°C and
`28°C) although some "cold-labile" proteins are effectively
`inactivated at low temperature (i.e., mitochondrial ATPase).
`Above 30 - 40°C, proteins vary widely in their stability, most
`becoming inactivated, but some remaining stable even upon
`boiling (i.e., bacterial alkaline phosphatase).
`
`23 of 148
`
`Fresenius Kabi
`Exhibit 1017
`
`

`

`Preparation for Protein Isolation
`
`21
`
`C. Storage
`
`As a rule, a protein's half-life is extended by storage at low
`temperatures. Whether the best storage conditions are at 4 °C, -
`20°Ct -80°C, or in liquid nitrogen (-200°C) depends on the protein
`and its intended use.
`
`For short-term storage (one day to one week), the protein can
`be stored at 4°C in solution if 1) functional activity (enzyme assay,
`ligand binding, etc.) does not decrease during this period, 2) no
`degradation is visible by 2-D electrophoresis (Chapter 7), and 3) no
`significant amount of pelleted material is obtained by centrifuging
`10 min at 20,000 x g (which would indicate denaturation).
`
`For long-term storage (more than one week), several options
`are available depending on the stabilit