`
`baKS.mneSerF
`Exhibit 1018
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`

`Methods in Enzymology
`
`Volume 182
`
`Guide to Protein Purification
`
`El>ITED BY
`
`Murray P. Deutscher
`
`DEPARTMENT OF BIOCJ:iEMISTRY
`UNIVERSITY OF CONNECTICUT HEALTH CBNTER
`FARMINGTON, CONNECTICUT
`
`ACADEMIC PRESS, INC.
`Harcourt Brace Jovanovich, Publishers
`San Diego New York Berkeley Boston
`London Sydney Tokyo Toronto
`
`2 of 142
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`
`

`

`Th.is book is prin!ed on acid-free paper. E)
`
`COPYRIGHT© 1990 BY ACADEMIC PRESS, INC.
`All Rights Reserved.
`No part of this publication may be reproduced or transmitted in any form or
`by any means, electronic or mechanical, including photocopy, recording, or
`any information storage and retrieval system, without permission in writing
`from the publisher.
`
`ACADEMIC PRESS, INC.
`San Diego, California 92101
`
`United Kingdom Ediiion published by
`ACADEMIC PRESS LIMITED
`24-28 Oval Road, London NW! 7DX
`
`LIBRARY OF CONGRESS CATALOG CARD NUMBER: 54-9110
`
`ISBN 0-12-182083-1
`(Hardcover)(alk. paper)
`ISBN 0-12-213585-7 (comb liound)(allc. pape()
`
`PRINTED IN ll!E UNITED STA'lcS OF AMERJCA
`9 8 1 6 5 4 3 2
`90 91 ll2 93
`I
`
`3 of 142
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`Fresenius Kabi
`Exhibit 1018
`
`

`

`[1]
`
`WHY l'U)UFY ENZVMES?
`
`1
`
`[1] Why Purify Enzymes?
`By ARTHUR KORNBERG
`
`uDon't waste clean thinking on dirty enzymes,. is an admonition of
`Efraim Racker's which is at the core of enzymology and good chemical
`practice. lt says simply that detailed studies of how an enzyme catalyzes
`the conversion of one substance to another is generally a waste of time
`unt.il the enzyme has been purified away from the other enzymes and
`substances that make up a crude cell extract. The mixture of thousands of
`different enzymes released from a disrupted liver, yeast, or bacterial cell
`likely contains several that direct other rearrangements of the starting
`material and the product of the particular enzyme's action. Only when we
`have purified the enzyme to the point that no other enzymes can be
`detected can we feel as~ured that a single type of enzyme molecule directs
`the conversion of substance A to substance B, and does nothing more.
`Only then can we learn how the enzyme does its work.
`The rewards for the labor of purifying an enzyme were laid out in a
`series of inspirational papers by Otto Warburg in the 1930s. From his
`laboratory in Berlin-Dahlem came the discipline and maiiy of the methods
`of purifying enzyme's and with those the clarification of key reactions and
`vitamin functions in respiration ahd the fermentation of glucose. War(cid:173)
`burg's contributions strengthened the classic approach to enzymology
`inaugurated with Eduard Biichner's accidental discovery, at the turn of
`this century, of cell-free conversion of sucrose to ethanol. One tracks the
`molecular basis of cellular function-alcoholic fermentation in yeast, gly(cid:173)
`colysis in muscle, luminescence in a fly, or the replication of DNA-by
`first observing the phenomenon in a cell-free system. Then one isolates
`the responsible enzyme (or enzymes) by fractionation of the cell extract
`and purifies it to homogeneity. Then one hopes to learn enough about the
`structure of the enzyme to explain how it performs its catalytic functions,
`responds to regulatory signals, and is associated with other- enzymes and
`structures in the cell.
`By a reverse approach, call it neoclassical, especially popular in re(cid:173)
`cent decades, one first obtains a structure and then looks for its function.
`The protein is preferably small and stable, and has been amplified by
`cloning or is commercially available. By intensive study of the protein and
`homologous proteins, one hopes to get some clues to how it functions. As
`the popularity of the neoclassical approach has increased, so has there
`
`METHODS tt;' ENZYMOLOGY, VOL. 182
`
`Copyright CJ 1990 by Academic Pre,s1 Inc.
`All rtgbts of reproduction in any Conn rescr;od,
`
`4 of 142
`
`Fresenius Kabi
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`

`

`2
`
`METHODS TN ENzyMOLOGY
`[11
`been a corresponding decrease in interest in the classical route: pursuit of
`a function to isolate the responsible structure.
`Implicit in the devotion to purifying enzymes is the faith of a dedicated
`biochemist of being able to reconstitute in a test tube anything a cell
`can do. In fact, the biochemist with the advantage of manipulating the
`medium: pH, ionic strength, etc., by creating high concentrations of
`reactants, by trapping products and so on, should have an easier time
`of it. Another article of faith is that everything that goes on in a cell is
`catalyzed by an enzyme. Chemists sometimes find this conviction difficult
`to swallow.
`On a recent occasion I was told by a mature and well-known physical
`chemist that what fascinated him•most in my work was that DNA replica(cid:173)
`tion was catalyzed by enzymes! This reminded me of a seminar I gave to
`the Washington University chemistry department when I arrived in St.
`Louis in 1953. I was describing the enzymes that make and degrade orotic
`acid, and began to realize that my audience was rapidly slipping away.
`Perhaps they had been expecting to hear about an organic synthesis of
`erotic acid. In a last-ditch attempt to retrieve their attention, I said loudly
`that every chemical event in the cell depends on the action of an enzyme.
`At that point, the late Joseph Kennedy, the brilliant young chairman.
`awoke: "Do you mean to tell us that something as simple as the hydration
`of carbon dioxide (to form bicarbonate) needs an enzyme?'' The Lord had
`delivered him into my hands. "Yes, Joe, cells have an enzyme, called
`carbonic anbydrase. It enhances the rate of that reaction more than a
`million fold.' '
`Enzymes are awesome machines with a suitable level of complexity.
`One may feel ill at ease grappling with the operations of a cell, let alone
`those of a multicellular creature, or feel inadequate in probing the fine
`chemistry of smalJ molecules. Becoming familiar with the personality of
`an enzyme performing in a major synthetic pathway can be just right. To
`gain this intimacy, the enzyme must first be purified to near homogeneity.
`For the separation of a protein species present as one-tenth or one-hun(cid:173)
`dredth of I% of the many thousands of other kinds in the cellular commu(cid:173)
`nity, we need to devise and be guided by a quick and reliable assay of its
`cataJytic activity.
`No enzyme is purified to the point of absolute homogeneity. Even
`when other proteins constitute less than 1 % of the purified protein and
`escape detection by our best methods, there are likely to be many millions
`of foreign molecules in a reaction mixture. Generally, such contaminants
`do not matter unless they are preponderantly of one kind and are highly
`active on one of the components being studied.
`
`5 of 142
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`

`

`[1]
`
`WHY PURIFY ENZYMES?
`
`1
`
`Only after the properties of the pure enzyme are known is it profitable
`to examine its behavior in a crude state. "Don't waste clean thinking on
`dirty enzymes" is sound dogma. I cannot recall a single instance in which
`I begrudged the time spent on the purification of an enzyme, whether it
`led to the clarification of a reaction pathway, to discovering new en(cid:173)
`zymes, to acquiring a Uilique analytical reagent, or led merely to greater
`expertise with purification procedures. So, purify, purify, purify.
`Purifying an enzyme is rewarding all the way, from first starting to free
`it from the mob of proteins in a broken cell to having it finally in splendid
`isolation. It matters that, upon removing the enzyme from its snug cellular
`niche, one cares about many inclemencies: high dilution in unfriendly
`solvents, contact with glass surfaces and harsh temperatures, and ex,po(cid:173)
`sure to metals, oxygen, and untold other perils. Failures are often attrib(cid:173)
`uted to the fragility of the enzyme and its ready denaturability, whereas
`the blame should rest on the scientist for being more easily denatured.
`Like a parent concerned for a child's whereabouts and safety, one cannot
`leave the laboratory at night without knowing how much of the enzyme
`has been recoveFed in that day's procedure and how much of the contami(cid:173)
`nating proteins still remain.
`To attain the goal of a pure protein, the cardinal rule is that the ratio of
`enzyme activity to the total protein is increased to the limit. Units of
`activity and amounts of protein must be strictly accounted for in each
`manipulation and at every stage. In this vein, the notebook record of an
`enzyme purification should withstand the scrutiny of an auditor or bank
`examiner. Not that one should ever regard the enterprise as a business or
`banking operation. Rather, it often may seem like the ascent of an un(cid:173)
`charted mountain: the logistics like those of supplying successively higher
`base camps. Protein fatalities and confusing contaminants may resemble
`the adventure of unexpected storms and hardships. Gratifying views
`along the way feed the anticipation of what will be seen from the top . The
`ultimate reward of a pure enzyme is tantamount to the unobstructed and
`commanding view from the summit. Beyond the grand vista and thrill of
`being there first, there is no need for descent, but rather the prospect of
`even more inviting mountains, each with the promise of even grander
`views.
`With the purified enzyme, we Learn about its catalytic activities and its
`responsiveness to regulatory molecules that raise or lower activity. Be(cid:173)
`yond the catalytic and regulatory aspects, enzymes have a social face that
`dictates crucial interactions with other .enzymes, nucleic acids, and mem(cid:173)
`brane surfaces. To gain a perspective on the enzyme's contributions to
`the cellular economy, we must also identify the factors that induce or
`
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`

`4
`
`METHODS IN ENZYMOLOGY
`
`{11
`
`repress the genes responsible for producing the enzyme. Tracking a meta(cid:173)
`bolic or biosynthetic enzyme_ uncovers marvelous intricacies by' which a
`bacterial cell gears enzyme production precisely to its fluctuating needs.
`Popular interest now centers on understanding the growth and devel(cid:173)
`opment of flies and worms, their cells and tissues. Many laboratories
`focus on the aberrations of cancer and hope that their studies will furnish
`insights into the normal patterns. Enormous efforts are also devoted to
`AIDS, both to the virus and its destructive action on the immune system.
`In these various studies, the effects of manipulating the cell's genome and
`the actions of viruses and agents are almost always monitored with intact
`cells and organisms. Rarely are attempts made to examine a stage in an
`overall process in a cell-free system. This reliance in current biological
`research on intact cells and organisms to fathom their chemistry is a
`modem version of the vitalism that befell Pasteur and that has permeated
`the attitudes of generations of biologists before and since.
`It baffles me that the utterly simple and proven enzymologic approach
`to solving basic problems in metabolism is so commonly ignored. The
`precept that discrete substances and their interactions must be under(cid:173)
`stood before more complex phenomena can be explained is rooted in the
`history of biochemistry and should by now be utterly commensensical.
`Robert Koch, in identifying the causative agent of an infectious disease,
`taught us a century ago that we _must first isolate the responsible microbe
`from all others. Organic chemists have known even longer that we must
`purify and crystallize a substance to prove its identity. More recently in
`history, the vitamin hunters found it futile to try to discover the metabolic
`and nutritional roles of vitamins without having isolated ·each in pure
`form. And so with enzymes it is only by purifying enzymes that we can
`clearly identify each of the molecular machines responsible for a discrete
`
`~
`
`,~
`
`ij :CAlif8ftf.U.A.;
`
`: 1
`
`PURIF'Y 1
`
`1
`
`FIG. 1. Personalized license plate expressing a commi1men1 10 enzymology,
`
`7 of 142
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`

`

`[1]
`
`WHY PURIFY ENZVMES?
`
`5
`
`metabolic operation. Convinced of this, one of my graduate students
`expressed it in a personalized license plate (Fig. 1).
`
`Acknowledgment
`
`This article .borrows extensively from ''For the Love of Enzymes: The Odyssey of ti
`Biochemist,'' Harvard University Press, 1989.
`'
`
`8 of 142
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`

`[2]
`
`GENERAL STRATEGIES AND CONSIDERATIONS
`
`9
`
`[2] Strategies and Considerations for Protein Purifications
`By STUART LJNN
`
`The budding enzymologist is generally surprised by the time necessary
`to develop a protein purification procedure relative to the time required to
`accumulate information once the purified protein is available. While there
`is no magic formula for desig,ning a protein purification, some forethought
`can help to expedite the tedious job of developing the purification scheme.
`This chapter is designed to point out some considerations to be under(cid:173)
`taken prior to stepping up to the bench. Once at the bench, the subsequent
`chapters of this book as well as two other recent publications concerning
`enzyme purification1•2 should serve as a guide.
`
`Preliminary Considerations
`
`What Is the Protein To Be Used For
`In these days of the biotechnology revolution, the required amount of
`purified protein may vary· from a few micrograms needed for a cloning
`endeavor to several kilograms required for an industrial or pharmaceuti(cid:173)
`cal application. Therefore, a very major consideration is the amount of
`material required. One should be aware of the scale-up ultimately ex(cid:173)
`tiected, and the final scheme should be appropriate for expansion to those
`levels. There are very real limitations to how far a procedure can be
`scaled up. These limitations are brought about not only by considerations
`of cost and availability of facilities, but also by physical constraints of
`such factors as chromatographic resin support capabilities and electro(cid:173)
`phoresis heating factors. As outlined below, individual steps of the proce(cid:173)
`dure should flow from high-capacity/low-cost techniques toward low-
`- capacity/high-cost ones. Nonetheless, in some cases two procedures may
`be required: for example, one to obtain microgram quantities for cloning
`and a second to produce kilogram amounts of the cloned material. The
`protein chemist should remain flexible for adopting new procedures when
`such changes are warranted.
`Another consideration is whether the protein must be active (an en(cid:173)
`zyme, a regulatory protein, or an antibody, for example), whether it must
`
`1 R. K. Scopes, "Protein Purification, Principles and Practice," 2nd Ed. Springer-Verlag,
`New York, 1987.
`1R. Burges, ed., "Pro_tein Purification, Micro to Macro." Alan R. Liss. New York, 1987.
`
`METHODS rN ENZ'iMOLOGY, VOL. 182
`
`Coi,;right C 1990 by Acad~mfo Pres>. Inc.
`Ali n5h1s of rerrnductlon in any fomi re.served.
`
`9 of 142
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`

`

`10
`
`DEVELOPING PURIFICATION PllOCEPURES
`
`[2]
`
`be in a native configuration, but .not associated with an activity, or
`whether it need not be in any specific configuration (a small peptide or a
`peptide to be utilized only for obtaining sequence information, for exam(cid:173)
`ple). The techniques employed should be as gentle as is necessary, but,
`whenever possible, some of the harsher but often spectacularly successful
`procedures such as those which involve extremes of pH, organic sol(cid:173)
`vents, detergents, or hydrophobic or strong affinity chromatographic me(cid:173)
`dia should also be used.
`
`Assays
`
`Possibly the most important preliminary step is to develop appropriate
`assays. The success of the purification is often most dependent on this.
`Five considerations come to mind: sensitivity, accuracy, precision, sub(cid:173)
`strate availability, and cost.
`Sensitivity is often the limiting factor as the protein becomes diluted
`into column effluents, etc. Before beginning a step, the likely dilution and
`losses ought to be estimated and the ability to detect the protein after a
`reasonably succ.essful procedure ought to be possible.
`Accuracy and precision are often compromised in these days of fast
`technology, but clearly these items must be controlled to the extent that
`the assay is reliable for assuring recovery of material and reproducibility.
`Specificity is usually a problem early in the purification. Often, how(cid:173)
`ever, substrates can be simplified or controls omitted as the purification
`progresses.
`Substrate availability and cost refer to the practicality of the assay:
`Can enough substrate be prepared to perform the entire purification with(cid:173)
`out interruption? Stopping to prepare more substrate or skimping on ma(cid:173)
`terial usually results in disaster. On the other hand, assays at certain steps
`in the purification might be modifiable, e.g. , leaving out specificity con(cid:173)
`trols at later stages or assaying alternate chromatography fractions.
`There is a recent trend not to use assays for protein activity, but to
`purify a gel band or an antigen instead. Although this tactic might be
`appropriate in instances where activity is not being sought, it is to be
`strongly discouraged when activity is in fact what is desired. It cannot be
`emphasized strongly enough that an activity assay is necessary to obtain
`optimal yields of activity, be it one associated with an enzyme, a DNA(cid:173)
`binding protein, an antibody~ or a hormone.
`A final comment pertains to the protein assay. Again, the goals are
`simplicity, reproducibility, specificity, and reliability. Accuracy is gener(cid:173)
`ally compromised, as no commonly used assay is absolute with regard to
`all proteins. With crude fractions, color reactions are probably best.
`
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`(2)
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`GENERAL STRATEGIES AND CONSIDERATIONS
`
`11
`
`TABLE I
`ADDITIONS TO PRbTI!lN SOLVENTS
`
`Class
`
`Examples
`
`Purpose
`
`Buffer
`Salt
`Detergents
`Su:rfactan1s
`Glycerol, sucrose
`
`Sodium azide
`Metal cheJators
`
`Sulfhydryl agents
`Ligands
`Protease inhibitors
`
`KCI, NaCl, (NH4)2SO•
`Deoxycholate
`Triton X-100
`
`EDTA (ethylenediaminetetraacetic acid),
`EGTA [ethylene glycol bis(,8-ami(cid:173)
`noethylether) N,N'-tetraacetic acid]
`2-Mercaptoethanol, dithiothreitol
`-M.g1+ , ATP, phosphate
`PMSF (phenylmethylsulfonyl fluoride),
`TPCK (N-tosyl-L-phenylalanine chloro(cid:173)
`methyl ketone), TLCK .(N•-p-tosyl-r..(cid:173)
`!ysine chloromethyl ketone)
`
`Stability
`Stability
`Stability, solubility
`Stability
`Stability; allows
`storage below o•
`in liquid state
`Bacteriostatic
`Stability
`
`Stability
`Stability
`Stability
`
`While the Bradford method3 is by far the simplest of these, in our labora(cid:173)
`tory we find it to be unreliable with crude fractions from animal cells or
`when detergents are present. For column effluents, ultraviolet absorption
`is optimal: it is sim_ple, sensitive, and does not consume the material. For
`extremes of sensitivity, wavelengths between 210 and 230 nm .can be
`utillzed.~•5 Again, protein assay procedures can and often must be
`changed as.the purification progresses.
`
`What Should Be Added to the Btiffers
`
`Once a purification scheme is developed, there is great resistance to
`modifying it, as modification requires laborious trial runs. The usual re(cid:173)
`sponse to "why is the protein suspended in x?'' is "iflleave it out, I don't
`know what will happen.'' The obvious lesson is to add something only
`with good reason in the first place.
`Solutes are added usually to improve stability I prevent th~ growth_ of
`microorganisms, reduce the freezing point-, or keep the protein in solu(cid:173)
`tion. Table I lists several classes and examples of such additions. It is well
`
`3 M. M. Bradford, Anal. Bi<Jchem . 12, 248 (1976).
`4 W, J. Waddell, J. Lab. Cli,1. Med. 48, 3ll (1956).
`s M. P. Tombs, F. Souter, and N . F . MacLagan, Biochem. J. 73, 167 (1959),
`
`11 of 142
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`

`12
`
`DEVELOPING PURIFICATION PROCEDURES
`
`[2]
`
`worth the effort to carry out stability studies (e.g., heat inactivation or
`storage trials) in order to learn how to maintain a stable protein. Two
`notes of caution: (1) optimal storage conditions change with purification;
`(2) optimal storage conditions need not relate to optimal conditions for
`activity. Indeed, additions which stabilize a protein often inhibit it when
`added to activity mixes. Of course, the latter situation must be considered
`when utilizing the protein-interfering substances will have to be re~
`moved or "diluted out" during utilization of the protein.
`In our experience, reducing agents are particularly effective with bac(cid:173)
`terial enzymes which derive from a reducing environment, whereas mam(cid:173)
`malian cell enzymes take kindly to surfactants and protease inhibitors.
`Fungal proteins also respond to protease inhibitors. Optimal pH and salt
`concentrations vary. Most enzymes prefer the lowest temperature allow(cid:173)
`able: 0° (on ice, not in a refrigerator) or -20° with glycerol or sucrose
`present. If frozen, storage above liquid nitrogen or at - 70° is often best.
`Special precautions which must be taken for purification and stabilization
`of large protein complexes· are noted in Section IX of this volume.
`A final note concerns the containers used for purified proteins or purifi(cid:173)
`cation fractions. Glass should not be used with very dilute solutions,
`plastic tubes being better. In our experience, polypropylene-based plas(cid:173)
`tics are superior to polyethylene ones, and polystyrene or other clear
`plastics are less satisfactory. Be sure to have tight-fitting caps if storage is
`in "frost-free" freezers.
`
`Co~taminating Activities
`
`Often proteins need not or cannot be obtained in a pure state, but
`particular interfering activities (e.g., nucleases, protease, phosphatases)
`must not be present. In our experience, attempting to pw·ify one activity
`against one or more others by doing multiple types of activity assays as a
`criterion of purity is an extremely frustrating endeavor. Instead, purifying
`so as to optimize yield and specific activity (units/mg protein) with selec(cid:173)
`tive choice of fractions only at the last or at most penultimate step is more
`likely to be satisfactory.
`
`Source of Protein
`
`When the source of a protein is not absolutely dictated, careful consid(cid:173)
`eration of the source is worth the time and effort, and trial extracts from a
`number of sources should be done.
`The cost and availability of the source, particularly if a largely scaled
`up preparation might be desirable in the future, should also be considered
`
`12 of 142
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`

`(2]
`
`GENERAL STRATEGIES AND CONSIDERATIONS
`
`13
`
`as well as the genetic knowledge and technology available for the organ(cid:173)
`ism should regulatory and/or gene sequence manipulations be envisioned.
`If the protein is to be overexpressed, is a bacterial or fungal cell better?
`Which one? What special precautions are necessary for each organism?
`Will the protein be appropriately processed?
`Once at the bench, several sources should be tested for.total yield of
`activity (per gram of starting material or per unit cost), the starting spe(cid:173)
`cific activity (units/mg protein), and the stability of the protein. In the
`extreme case, the classical microbiological approach of isolating micro(cid:173)
`organisms with unique growth requirements might lead to unexpected
`success.
`
`Preparing Extracts
`
`Preparing extracts is discussed in Section IV of this volume so only
`general considerations will be noted here. In our experience, ·the manner
`in which cells are disrupted has a profound and unpredictable effect on
`the yield and quality of the protein preparation. Trials are clearly ney
`essary.
`Thought should always be given toward scaling up the preparation,
`and how the disruption procedure will or will not adapt to being scaled up.
`Will the volumes or time required become unreasonable? Can a subse(cid:173)
`quent clarification step also be conveniently scaled up?
`In general, volumes should be kept as small as possible, i.e., extracts
`as c.oncentrated as possible. Tissue, cell type, or organelle fractionation is
`almost always worthwhile prior to disruption. Finally, consideration
`should also be given to the substance in which the starting material is
`suspended so that the protein desired is ~oluble and/or stable. Of course,
`the contents of the suspension buffer should not interfere with the subse(cid:173)
`quent step(s) in the purification procedure.
`
`Bulk or Batch Procedures
`
`These procedures are almost always utilized early in the purification
`as they are often most effective in removing nonprotein material and are
`most amenable to the large volume and amounts of material that exist in
`earlier stages of the preparation. A great deal of effort went into designing
`these steps in the early days of protein chemistry, and much frustration
`can probably be avoided by reinstituting some of these old-fashioned
`procedures.
`- Section Vl of this volume outlines some of these approaches. Drastic
`methods such as heat, extremes of pH, or phase partition with organic
`
`13 of 142
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`

`

`14.
`
`DEVELOPING PURJF[CATION PROCEDURES
`
`[2)
`
`solvents might be particularly effective with stable proteins, though subtle
`forms of damage may be difficult to foresee or to detect. Gentler proce(cid:173)
`dures include phase partition with organic polymers, "salting out," or.
`addition of ion-exchange resin as a slurry. Batch elution from large, high(cid:173)
`capacity ion-exchange columns might also be effective. The time ex(cid:173)
`pended in developing and optimizing these early steps is always worth(cid:173)
`while-even a factor of two increase in specific activity may decide the
`feasibility of a subsequent step from both cost and technical consider(cid:173)
`ations.
`
`Refined Procedures
`
`Once the bulk methods have yielded a protein preparation which is
`reasonably free of nucleic acids, polysaccharides, and lipids, the prepara(cid:173)
`tion becomes amenable to the more interesting and spectacular proce(cid:173)
`dures which have been developed in recent years. The general strategy is
`to proceed from high- to low-capacity procedures and to attempt to ex(cid:173)
`ploit specific affinity materials whenever possible.
`Applications and technical details for these procedures are noted in
`Sections VII, Vill, IX, and XI of this volume, and will not be described
`here beyond citing examples. As a general consideration, in proceeding
`from one procedure to the next, one ought to reduce as much as possible
`the necessity for dialysis and concentration. Hence, procedures that sepa(cid:173)
`rate by size can also be exploitei•to remove salt. Procedures utilizing
`high-capacity resins can concentr~te proteins as well as purify them, or
`resins from which proteins elute at low-salt concentrations can be directly
`followed with resins to which the protein binds at higher salt concentra(cid:173)
`tions. Also, some steps (e.g., sedimentation through gradients of sucrose
`or glycerol) may leave the protein in a medium which might be ideal for
`long-term storage, but difficuJt (or appropriate) for utilization in a subse(cid:173)
`quen1 step. Finally, interchanging the order of the steps of a procedure
`can, and often does, have a profound effect on the success of a purifica(cid:173)
`tion scheme.
`Some procedures which cannot be effectively scaled up [e.g., sedi(cid:173)
`mentation, or higb-\'erformance liquid chromatography (HPLC)] can be
`carried out with ·small aliquots of the preparation, but only if left to the
`final stages. (In some instances the utilization of aliquots is desirable, the
`less purified fractions may be more stable to long~term storage.)
`
`High-Capacity Steps
`Generally, these include ion-exchange resins or very general affinity
`agents such as dyes or glass. When used for large amounts of material;
`
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`

`

`[2)
`
`15
`GENERAL STRATEGIES AND CONSIDERATIONS
`ioo-exchange resins can often be successfully reutilized at a later stage for
`additional purification (especially if the pH is changed) or for concen(cid:173)
`tration.
`
`Intermediate-Capacity Steps
`These might include the hydrophobic resins for which long chromato(cid:173)
`graphic times reduce activity yields. Many affinity agents (bulk DNA or
`simple DNA sequences, immunoaffinity, or ligands of a protein) fall into
`this class. In these instances, thought and effort must be given to finding
`materials that can successfully elute the protein without destabilizing or
`inactivating it. Gel filtration should also be considered as a step with
`intermediate capacity.
`
`Low-Capacity Steps
`Affinity steps utilizing valuable ligands such as substrate analogs,
`complex DNA sequences, and lectins might be included here. Also in(cid:173)
`cluded are isoelectiic focusing (precipitation is often a problem with mod(cid:173)
`erate amounts of protein), electrophoresis, HPLC (which in our bands is
`difficult to scale up without loss of resolution), and ultracentrifugation.
`Very small hydrophobic columns might also be successful where larger
`ones have failed.
`
`Conclusions
`Though protein purification is often a difficult and frustrating process,
`its rewards are great. Moreover, with the continual development of new
`technology, the commercial availability of materials utilized for purifica(cid:173)
`tion procedures, and the availability of genetically altered sources of ma(cid:173)
`terial, the future bodes well for simpler procedures accompanied by
`greater rewards and indeed for protein chemistry as well.
`
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`

`24
`
`GENERAL METHODS FOR HANDUNG PROTEINS AND ENZYMES
`
`[4]
`
`[ 4] Buffers: P rinciples and Practice
`By VINCENT s. STOLL and JoHN s. BLANCHARD
`
`The necessity for maintaining a stable pH when _studying enzymes is
`well established. 1 Biochemical processes can be severely affected by mi(cid:173)
`nute changes in hydrogen ion concentrations. At the same time many
`protons may{be consumed or released during an enzymatic reaction. It
`has become increasingly important to find buffers to stabilize hydrogen · •
`ion concentrations while not interfering with the function of the enzyme
`being studied. The development of a series of N-substituted taurine and
`glycine buffers by Good et al. has provided buffers in the physiologically
`relevant range (6.1-10.4) of most enzymes, which have limited side ef(cid:173)
`fects with most enzymes.2 It has b~en found that these buffers are 0011-(cid:173)
`toxic to cells at 50 mM concentrations and in some cases much higher. 3
`
`Theory
`
`The observation that partially neutralized solutions of weak acids or
`weak bases are resistant to pH changes on the addition of small amounts
`of strong acid or strong ·base leads to the concept of• 'buffering''. 4 Buffers
`consist of an acid and its conjugate base, such as carbonate and bicarbon(cid:173)
`ate, or acetate and acetic acid. The quality of a buffer is dependent on its
`buffering capacity (resistance to change in pH by addition of strong acid
`or base), and its ability to maintain a stable pH upon dilution or addition of
`neutral salts. Because of the following equilibria, additions of small
`amounts of strong acid or strong base result in the removal of only small
`amounts of the- weakly acidic or basic species; therefore, there is little
`change in the pH:
`
`HA (-acid) ~ H + + A - (conjugate base)
`B (base) + H · ~ BH+ {conjugate acid)
`
`(I)
`(2)
`
`The pH of a solution of a weak acid or base may be calculated from the
`Henderson-Hasselbalch equation:
`
`1 R. J. Johnson and D. E. Metzler, this series, Vol. 22, p. 3; N. E. Good and S. lzawa. Vo!.
`24, p. 53_
`i N. E. Good, G.D. Winget, W. Winter, T.N. Connolly, S. lzawa, and R. M. M. Singh,
`Biochemistry S, 467 (1966).
`' W. J. Ferguson et al., Anal. Biochem. 104, 300 (1980),
`'D. D. Perrin and B. Dempsey, ''Buffers for pH and Metal Ion Control." Chapman &ff all,
`London, 1974.
`
`METHODS IN ENZYMOLOGY, VOL. 182
`
`Copyright O 1990 by Acnd<mio Pres,, lnc.
`All rights of reproduction in any form reserved,
`
`16 of 142
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`25
`
`(3)
`
`(4)
`
`BUFFERS: PRINCIPLES AND PRACT[CE
`pH = pK~ + log[basic species]/[aciclic species]
`The p/Ca_ of a buffer is that pH where the concentrations of basic and acidic
`species are equal, and in this basic form the equation is accurate between
`the p