Practical Protein Chemistry - A. Darbre 1989

Instead of an Introduction

I continue to experience a deep sense of satisfaction regarding the initiative taken by Dr. Darbre in bringing together a group of authors to unite all protein analytical chemistry methodology within a single volume. This endeavor appears all the more timely since the advent of Introduction/32.html">Genetic Engineering in general, and Protein Engineering in particular, has substantially elevated The Role of specialists in Protein Chemistry. Of course, it is now quite evident that the most efficient METHOD FOR DETERMINING Amino acid sequences relies on DNA Sequencing of a cloned Gene, or cloned DNA (cDNA). Furthermore, the fastest and most reliable approach to cloning involves screening gene or cDNA banks using oligonucleotide probes of minimal degeneracy, synthesized on The basis of carefully selected peptide fragments. In addition, only a few genes are expressed directly without post-translational Processing, solely As a result of proteolysis and/or amino acid side-chain modification; consequently, researchers must be able to identify evidence of modification and processing in the native product. Even when lacking a profound scientific objective, it is simply gratifying to "have in hand" peptide fragments corresponding to the coding sequence incorporated into DNA.

For many of us who witnessed the early days of molecular biology, protein engineering symbolizes the pinnacle of collaboration that molecular geneticists, protein chemists, and crystallographers have striven for. The diagram in Fig. 1 provides a General Overview of the collaborative pathways between the chemist and the genetic engineer, while the diagram in Fig. 2 illustrates the areas in which the chemist works in partnership with the crystallographer. Together, such a team can undertake the elucidation of the principles governing polypeptide chain folding or the Mechanism of Enzymatic catalysis, ultimately making it possible one day to theoretically translate a gene Structure into primary, and subsequently tertiary, Cell/13.html">Protein Structure, and even to predict the catalytic Properties of the final product.

However, today even a talented protein chemist, inspired by these unfolding Prospects, faces A number of challenges somewhat reminiscent of the "dark ages" of protein chemistry, when peptide Cleavage Methods, purification techniques, and sequencing protocols were non-existent, and everyone was consequently forced to invent their own approaches. Today, the sheer Abundance of methods is almost intimidating, requiring researchers to carefully select only those techniques best suited to solving a specific problem. To some extent, this very circumstance dampens the influx of young scientists into the field.

Literature data indicate that protein sequencing is nowadays performed using automated systems operated by technicians trained in handling vacuum lines. The protein chemist is convinced (and with some justification) that their scientific interests should lie somewhat elsewhere—specifically, in a profound understanding of amino acid chemistry and the prediction of property changes accompanying the perturbations of polypeptide chain folding. Such a scientist finds aesthetic satisfaction in designing a simple yet elegant experiment to solve a concrete problem, while automated analyzers, sequencers, and mass spectrometers, though necessary attributes of their work, essentially serve merely as tools to accomplish scientific objectives.

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FIG. 1.

FIG. 2.

Ultimately, the publication of this book should be warmly received by specialists, as alongside the inevitable wealth of detailed "recipes," it includes sound practical advice. However, the book will likely prove most valuable to the general biochemist who suddenly finds themselves needing to work in protein chemistry, for which they require a single vade mecum* that has not existed until now. Now, they have it.

Director of the Biotechnology Centre,

Royal College of Science and Technology, London

B. S. Hartley

* Vade mecum — from Latin *vade mecum*, meaning "go with me." This is the established term for handbooks and guidebooks, particularly of small format. — Transl. note.

SOME Abbreviations AND DESIGNATIONS USED IN THE BOOK

APS — aminopropyl Glass

ß-APS — ß-N-aminoethyl-(3-aminopropyl) glass

ATZ — 2-anilino-5-thiazolinone

FPLC — fast protein liquid Chromatography

BSA — bovine serum albumin

IBDAB — bis-(1,1-trifluoroacetoxy)iodobenzene

BAWP — butanol — acetic acid — Water — pyridine

HPLC — High-Performance Liquid Chromatography

HETP — height equivalent to a theoretical plate

GLC — Gas-Liquid Chromatography

GP — gas-phase

HFBA — heptafluorobutyric acid

DABITC — 4-dimethylaminoazobenzene-4'-isothiocyanate

DABTH — 4-dimethylaminoazobenzene-4'-thiohydantoin

HPFAE — 2-(hydroxypropyl)aminoethyl

DITC — p-phenylene diisothiocyanate

DMAA — dimethylallylamine

DMBA — dimethylbenzylamine

DMSO — dimethyl sulfoxide

DMF — dimethylformamide

DNS — 5-dimethylaminonaphthalene-1-sulfonyl

DNP — dinitrophenyl

DOC — sodium deoxycholate

SDS — sodium dodecyl sulfate

DTNB — 5,5'-dithiobis(2-nitrobenzoic acid) — Ellman's reagent

DTT — dithiothreitol

DCC — dicyclohexylcarbodiimide

DFP — diisopropyl fluorophosphate

DEAE — diethylaminoethyl OD units — optical density units

LFP — liquid-phase

IEC-HPLC — ion-exchange high-performance liquid chromatography

quadrol — N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine

CM — carboxymethyl

MITC — methyl isothiocyanate

NBF — 4-chloro-7-nitrobenzofurazan

NMM — N-methylmorpholine

DTNB — 5,5'-dithiobis-(2-nitrobenzoic acid)

NTSB — 2-nitro-5-sulfothiobenzoate

NTCB — 2-nitro-5-thiocyanobenzoate

NEM — N-ethylmorpholine

OPA — o-phthalaldehyde

RP-HPLC — reversed-phase high-performance liquid chromatography

PAGE — polyacrylamide gel

PMA — pyromellitic acid Polybrene — 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide

PFB-Br — 2-bromo-2,3,4,5,6-pentafluorotoluene

SP — sulfopropyl

THF — tetrahydrofuran

TEMED — tetramethylethylenediamine

TMA — trimethylamine

TNBS — 2,4,6-trinitrobenzenesulfonic acid

TNP — 2,4,6-trinitrophenyl

Tris — tris(hydroxymethyl)aminomethane

SP — solid-phase

TPCK — L-(1-tosylamido-2-phenylethyl) chloromethyl ketone

TFA — trifluoroacetic acid

TEA — triethylamine

TETA — triethylenetetramine

FITC — phenyl isothiocyanate

PMSF — phenylmethylsulfonyl fluoride

PTH — phenylthiohydantoin

PTC — phenylthiocarbamoyl

P — phospho

EDC — 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

Boc — tert-butyloxycarbonyl

BNPS-skatole — 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine

CPG — controlled-pore glass

HNBBr — 2-hydroxy-5-nitrobenzyl bromide

HOBt — 1-hydroxybenzotriazole

HOSu — N-hydroxysuccinimide

NBS — N-Bromosuccinimide