Redox-active amino acids in biology

450 Pages·1995·19.3 MB·English

by  Colowick Melvin

Preface
Recent advances in enzymology have dramatically altered our knowl-
edge of cofactor catalysis. In contrast to the classical picture, whereby
cofactors are viewed as freely dissociating low-molecular-weight com-
pounds, an increasing number of enzyme systems have been documented
as using peptide-bound amino acid side chains as catalytic units in redox
chemistry. As described here, these new prosthetic groups may be either
radicals derived from glycyl, tyrosyl, and trypyophanyl residues or quinones
derived from the posttranslational oxidation of tyrosyl or tryptophanyl
precursors. Although the term rotcafoc has traditionally been reserved for
small molecules, I propose that we extend this terminology to include the
new functional groups derived from amino acid side chains.
The goal of this volume is to introduce the researcher to the methods
available for the detection and characterization of amino acid side chains
functioning as redox catalysts. Although the field is moving rapidly and
new structures may emerge in the near future, the chapters contained herein
should provide the groundwork for anyone wishing to enter this exciting
field. I am indebted to all of the contributors for their willingness to submit
their chapters in a timely manner; to John Abelson and Mel Simon for
their original interest in the volume; and to Shirley Light for her skill and
persistence in producing a final book.
HTIDUJ NAMNILK
ix
1] [ GNINIATNOC-APOD SNIETORP 1
Quinone
[ 1] Precursors of Tanning:
Dopa-Containing Proteins
By J. HERBERT WAITE
Introduction
Dopa-containing proteins are proteins that contain the amino acid 3,4-
dihydroxyphenyl-L-alanine in their primary sequence. They appear to be
rather widely distributed in nature and have been reported in at least
five different animal phyla. 1 Although the functions attributed to dopa-
containing proteins are still speculative, they are commonly associated with
extraorganismic structural materials such as helminth egg cases, 2 molluscan
byssus, 4'3 annelid cement, 5 and ascidian tunic. 6 Dopa proteins are usually
cosecreted with stoichiometric quantities of the enzyme catechol oxidase
(EC 1.10.3.1) 1 which is known to catalyze the oxidative dehydrogenation
of peptidyl-dopa groups to peptidyl-dopaquinones. There is little agreement
about what follows the generation of the quinones. Suffice it to say for the
present that the appearance of the quinones leads to a rapid discoloration
and sclerotization of the proteins, rendering them intractable to all but
hydrolytic treatments. The term "quinone tanning" has been attached to
describe this chemical and physical transformation. Many have argued that
quinone tanning results from the formation of quinone-derived intermolec-
ular cross-links, 7 but this point has so far eluded biochemical verification/
Comparison of dopa protein sequences with their corresponding cDNA-
derived sequences suggests that peptidyl-dopa owes its existence to the
action of putative co- or posttranslational modifications on peptidyl tyro-
sines. 9-u Nothing is known about these putative hydroxylases except that
1 j. H. Waite, Comp. Biochem. Physiol. B 97B, 91 .)0991(
2 j. H. Waite and A. .M Rice-Ficht, Biochemistry ,62 7819 .)7891(
3 j. H. Waite, .J Biol. Chem. 2911 258, .)3891(
L. 4 .M Rzepecki, .K .M Mueller, and .J H. Waite, Biol. Bull. (Woods Hole, Mass.) 183,
321 .)2991(
5 j. H. Waite, R. A. Jensen, and D. E. Morse, Biochemistry ,13 5733 .)2991(
L. 6 .C Dorsett, .C J. Hawkins, .J A. Grice, .M F. Lavin, P. .M Merefield, D. L. Parry, and
I. L. Ross, Biochemistry ,62 8078 .)6891(
R. Davies v and J. L. Frahn, .J Chem. Soc., Perkin Trans. ,1 p. 2295 .)7791(
8 .S .M Schaefer, J. and Waite, H. Hansen, J. D. Holl, Arch. Biochem. Biophys. 302, 552 .)3991(
D. R. 9 Filpula, S.-M. Lee, R. .P Link, .S L. Strausberg, and R. L. Strausberg, Biotechnol.
Prog. ,6 171 .)0991(
01 R. A. Laursen, Results Prob. Cell Differ. ,91 55 .)2991(
Copyright © by 1995 Academic Press, Inc.
METHODS IN ENZYMOLOGY, VOL. 852 All rights of reproduction in any form reserved.
2 EVITCA-XODER ONIMA NI SDICA YGOLOIB [ ]1
the hydroxylation may be autocatalytic. 21 Because of the wide variety of
compositions, primary sequences, and molecular weights in the dopa-con-
taining proteins, there is no single purification strategy that applies to all
such proteins. There are, however, two caveats that apply generally to
working with these proteins:
.1 Dopa is oxidatively unstable at physiological pH and above. 31 It is
therefore critical to work at low pH whenever possible; when a pH > 7
is necessary, use borate (which complexes dopa) or an antioxidant such
as ascorbateJ 4
2. The diphenolic moiety of dopa has an extremely high affinity for
metals, particularly iron(III) and aluminum(III). This applies whether the
metal atom is present as a cation, oxide, mineral, or metal. 71-51 Avoid
exposing the protein to metals if dopa recovery is desired.
The purpose of this chapter is to outline some useful methodology for
the isolation and characterization of two different groups of dopa proteins:
(A) proteins with tandemly repeated sequences from the common mussel
Mytilus edulis, and (B) protein precursors of the eggshells of the liver fluke
Fasciola hepatica. Some techniques with regard to dopa-containing proteins
have been previously described in this series is and will not be repeated
here, but rather referred to whenever pertinent.
Procedures for Extracting and Isolating Dopa-Containing Proteins
Extraction of M. edulis Foot Proteins
Dopa-containing byssal precursor proteins are stockpiled in the mussel
foot, a small pigmented appendage in the center of the animal. The location
of the proteins has led to the name "Mytilus edulis foot proteins" and the
acronym Mefp. Any plans for the preparation of dopa-containing Mefps
must include a mussel shucking party for the purpose of accumulating a
100 or more mussel feet which can be stored indefinitely at -80 .° M. edulis,
11 A. .C Rice-Ficht, .K A. Dusek, G. J. and Kochevar, J. H. Waite, .mehcoiB .loM .lotisaraP
,45 921 .)2991(
21 A. /~berg, .M Orm6, .P Norlund, and B.-M. ,grebOjS yrtsimehcoiB ,23 5489 .)3991(
31 Duleba, A. Sarna, T. .W H. and Korytowski, ,ztrawS .hcrA .mehcoiB .syhpoiB 041,002 .)0891(
41 j. H. Waite, Anal .mehC ,65 5391 .)4891(
51 .M .B McBride and .L G. Wesselink, .norivnE .icS .lonhceT ,22 307 .)8891(
61 .S .W Taylor, .C J. and Hawkins, D. J. Winzor, .gronI Chem. ,23 224 .)3991(
71 .V .K .F Chia, .M .P Soriaga, A. .T Hubbard, and .S E. Anderson, .J Phys. Chem. ,78
232 .)3891(
81 j. H. Waite and .C .V Benedict, this series, Vol. ,701 .p .793
11 DOPA-CONTAINING PROTEINS 3
colloquially called "blue," "common," or "sea" mussels, should have a
shell length of at least 4 cm and can be obtained either by collection from
the rocky intertidal zone or from seafood wholesalers. The latter course is
recommended for neophytes. Mussels obtained from a wholesaler should
be examined for vigor before use. The "litmus test" for mussel vigor is a
simple one based on gaping. When mussels are maintained out of sea water,
they gape, i.e., their valves are ajar. If they close their valves promptly when
tapped or pinched, then they are in fine fettle. If not, they are moribund and
should be discarded. If mussels are to be obtained by collection from the
field, some care is required to sort the different species that can coexist in
natural clusters. This is particularly true of the U.S. west coast where as
many as three species can occur in coaggregated clusters.
The following procedure has been jointly developed by laboratories in
the United States and Chile. °2,91 It appears to be applicable to the extraction
of homologous proteins from a variety of marine mussel species. 91 On
occasion, however, it has been necessary to "tweek" the acetone concentra-
tion used to precipitate efficiently the target protein(s). This was the case
for dopa protein from the hairy mussel Trichomya 91.atusrih The procedure
does not work for byssal precursor proteins from the zebra mussel Dreissena
polymorpha. 12
Materials A
0.7% (w/v) perchloric acid (PCA) cooled to 4 °
Ethanol-water ice bath
Concentrated sulfuric acid
Acetone cooled to -80 °
5% acetic acid cooled to 4 °
Equipment: Waring blender, deep freezer, refrigerated preparative
centrifuge, magnetic stirrer, small tissue grinder (Kontes, Vine-
land, N J)
Procedure A. The following protocol for isolating Mepf-1 and -2 can
be scaled according to the wet weight of tissue to be extracted. About 20
to 30 g of feet (wet weight) can be obtained from 100 mussels depending
on size. About 30 g feet are placed in 300 ml ice-cold 0.7% PCA and
homogenized for 1 min to a smooth puree using a small Waring blender
at top speed. The puree is centrifuged at 30,000g for 30 rain, and the
supernatant (S-l) is carefully decanted and its volume recorded. S-1 is
9a L. M. Rzepecki, X.-X. Qin, J. H. Waite, and M. F. Lavin, Mar. MoL Biol. Biotechnol. 1,
123 (1991).
02 j. Pardo, E. Gutierrez, C. Saez, M. Brito, and L. O. Burzio, Protein Express. Purif. 1,
147 (1990).
12 L. M. Rzepecki and J. H. Waite, Mar. Mol. Biol. Biotechnol. 2, 267 (1993).
4 REDOX-ACTIVE AMINO ACIDS IN BIOLOGY 1
transferred to a l-liter beaker, chilled in an ethanol-water ice bath (>0°),
and vigorously stirred on a magnetic stirrer. While stirring, concentrated
sulfuric acid is added (vol = 1-S × 0.0168) to give a final concentration of
3.0 M. This is followed by the dropwise addition of prechilled acetone
(2 × 1-S vol) using a large separatory funnel. When all the acetone has
been added, the suspension is left to sit for 15-20 min. After redispersing
the precipitate with gentle stirring, it is centrifuged for 03 min at 30,000g
as before. Do not use polycarbonate centrifuge tubes with the acetone-
containing supernatants. The second supernatant is decanted and can be
discarded in most cases. The pellets are left to drain upside down on
ice and are then resuspended by 5.0 ml vol of %5 acetic acid (S-3) and
rehomogenized in small (2 ml) tissue grinders (Kontes, Vineland, N J). The
samples are spun at 15,000g for 5 min in a microfuge, and are then ready
for further purification by gel filtration and high-performance liquid chro-
matography (HPLC).
Extraction of ViteUine Proteins from the Liver Fluke
Liver flukes (F. hepatica) are hermaphroditic, continually active sexu-
ally, and prolific egg layers producing some 30,000 eggs per day in sheep. 22
The eggs are protected by an organic shell or varnish that is derived from
proteins produced in the vitelline gland network. For obvious reasons, the
vitelline gland-extracted proteins are called "vitelline proteins" or Vps. 22 In
some parts of the world, e.g., southwest United States, liver fluke infection of
livestock can reach epidemic proportions. Public slaughterhouses in these
regions are the best places to collect liver flukes. Freshness of material is
absolutely essential when working with trematodes. This is in part due to
the profusion of proteolytic enzymes in the flukes. Even a few minutes at
room temperature can lead to a significant autolysis of animal tissues.
Materials B
Neutral salt buffer: 0.15 M NaC1, 0.05 M Tris(hydroxymethyl)amino-
methane hydrochloride (Tris-HCl), pH 7.5, with 1 mM phenylmeth-
ylsulfonyl fluoride, 01 mM N-ethylmaleimide, 52 mM ethylenedi-
aminetetracetic acid (EDTA), and 1 mM potassium cyanide
%5 (v/v) acetic acid with 4 M urea, 01 /xM leupeptin, and 1 mM
iodoacetamide chilled to 4 °
%5 acetic acid with 8 M urea chilled to 4 °
2.5% acetic acid chilled to 4 °
Equipment: Deep freezer, refrigerated preparative centrifuge, large
tissue grinders (Kontes), magnetic stirrer
22 j. H. Waite and A. M. Rice-Ficht, yrtsimehcoiB 28, 6104 (1989).
1 GNINIATNOC-APOD SNIETORP 5
Procedure B. The following can be used to purify dopa-containing vitel-
line proteins B and C: Worms collected from condemned livers at the
abattoir are immediately dropped into liquid nitrogen for transport and
storage. Prior to extraction, 15- to 20-g (wet weight) worms are briefly
thawed, spread out on 5-mm-thick glass plates, and deep frozen at -80 .°
The plate strategy allows for the selective dissection of the vitellaria from
the rest of the animal. 22 The vitellaria contain the eggshell precursors and
are located in the posterior third and peripheral 3-5 mm of the animal,
The dissective separation of the vitellaria from the more interior digestive
diverticulum is advisable to minimize protein degradation (Fig. 1). Once
dissected, the vitellaria are homogenized in large frosted glass tissue grind-
ers using the neutral salt buffer (10 ml/g wet weight). The homogenate is
centrifuged at 5000g at 4 ° for 10 min, and pellets are collected, rehomoge-
nized in neutral salt buffer, and centrifuged as before. The second pellet
is carefully collected and rehomogenized in cold 5% acetic acid with 4 M
urea and inhibitors (5 ml/g wet weight). The inhibitors, particularly leupep-
tin, are indispensible for the efficacy of this step. 22 If, in time, yield of the
dopa protein decreases, it is quite possible that the leupeptin has begun to
deteriorate. Homogenates are centrifuged at 35,000g at 4 ° for 30 rain. On
collection, supernatants are either stored frozen at -20 ° or brought to 10%
(w/v) ammonium sulfate at room temperature, stirred an additional 40 min
at 10 °, and allowed to sit without stirring for 10 min. The precipitate is
removed by centrifugation at 5000g for 30 min. The ammonium sulfate
concentration is then raised to 20% (w/v) at room temperature and stirred
as before. The precipitate is removed again by centrifugation. The superna-
tant is collected and dialyzed against at least 200 vol of 2.5% acetic acid at
f
, I
.GIF l. A whole mount of aloicsaF stained with Arnow's reagent 32 to ezilausiv the .airalletiv
sehsaD denote the approximate line of .noisicni Only the hcir-enilletiv lower third si retained.
The scale bar si equal to 1 .mc
6 REDOX-ACTIVE AMINO ACIDS IN BIOLOGY [ ]1
TABLE I
PURIFICATION TABLE ROF Mytilus edulis FOOT PROTEIN NO. 1 (Mefp-1) BASED NO EHT
ARNOW DNA BRADFORD DETECTION METHODS ROF DOPA DNA PROTEIN, YLEVITCEPSER
Total protein Total dopa Dopa/protein Yield Fold
petS )gm( (gmol) (/zmol/mg) )%( noitacifirup
From 53 g of feet
S-l, perchloric acid 221.0 75.0 0.34 001 0.1
S-3, acetone ppt 2.07 43.8 0.62 85 2.1
Sephadex G-200 30.9 1.23 40.1 43 1.3
C-8 HPLC 8.31 2.61 71.1 12 5.3
4 `° using dialysis tubing with a molecular weight cutoff of 1000 (Spectrum
Industries, Los Angeles, CA). Upon equilibration, protein in the tubing
flocculates and can be harvested by gentle centrifugation at 10,000g for
30 min in a swinging bucket rotor. Centrifuge tubes can be drained upside
down after discarding the supernatant, and the precipitate at the bottom
can be redissolved in a small volume of 5% acetic acid with 4-8 M urea.
It should be vigorously microfuged (5 min at 15,000g) before purification
by HPLC.
Methods for Detection of Dopa Proteins
Enzyme purifications are commonly evaluated by changes in specific
activity to ascertain yields and folds of purification. This can be applied to
dopa-containing proteins using specific assays for dopa and protein and
determining a dopa/protein ratio. 91'5 The assay of choice for dopa is that of
Arnow 32 described previously in this series, is For protein, it is the Bradford
assay 42 available as a kit from Bio-Rad (Richmond, CA). Table I itemizes
typical dopa/protein ratios for the purification of Mefp-1. Two features are
notable: the apparent yield is low, and the fold purification is little more
than three times that of the PCA extract. The apparent yield is low because
there are several different dopa-containing proteins extracted by PCA.
Mefp-2, in fact, is much more abundant than Mefp-1. When it is removed
from the latter by chromatography on Sephadex G-200, the total dopa level
decreases sharply. Fold purification is low because PCA is very selective
in that only a few proteins are extracted to begin with. If the dopa/protein
ratio of the intact mussel tissue could be estimated, it would likely be a
small fraction of the PCA "specific activity." Finally, it is important to
32 L. E. Arnow, .J Biol. Chem. 118, 531 (1937).
42 M. M. Bradford, Anal. Biochem. ~27 248 (1976).
1 DOPA-CONTAINING PROTEINS 7
1 2 3 4 1 2 3 4
<1
<2
Protein N BT
Fie. 2. Acetic acid-urea polyacrylamide gel electrophoresis of samples taken from different
steps in the purification of Mefp-1. The protein panel was stained with Coomassie blue R-
250, whereas the NBT panel was stained with nitroblue tetratrazolium in glycinate. Lane ,1
10/zg HPLC-purified protein; lane 2, 10/zg first peak Sephadex G-200; lane 3, 10/~g of S-3;
and lane 4, 10 tzg of S-1. See Table I for dopa/protein ratios. Arrows denote positions of
Mefp-1 and -2.
point out that since there are often several different kinds of dopa-con-
taining proteins in the same tissues, the dopa/protein ratio, while useful,
cannot be relied upon per se to reflect the degree of purity. Purity can,
however, be assessed when dopa/protein ratios are coupled to results fol-
lowing electrophoresis on acid-urea polyacrylamide gels. The application
of gel electrophoresis to dopa-containing proteins has been described earlier
in this series. In short, a purification strategy would entail sampling each
MT
critical step, e.g., those listed in Table I, by acid-urea polyacrylamide gel
electrophoresis. Proteins can be detected by staining with Coomassie blue
R-250 and dopa, by a modification of the Arnow assay, or by redox
MT
cycling 52 (Fig. 2). Redox cycling reactions involving nitroblue tetrazolium
(NBT) and glycinate are not as specific for o-diphenols as the Arnow
reaction, but they are much more sensitive and produce a more stable
color. 62 Acid-urea gels can be quickly and directly stained with NBT. (Sigma
Chemical Co.) as follows: Prepare 1 liter of 2 M glycinate (sodium salt),
52 M. A. Paz, R. Fltickinger, A. Boak, H. Kagan, and P. M. Gallop, J. Biol. Chem. 266,
689 (1991).
62 R. Fltickiger, M. A. Paz, and P. M. Gallop, this volume 11.
8 REDOX-ACTIVE AMINO ACIDS IN BIOLOGY 1
pH .01 Dissolve 5 mg of NBT in 1 ml of methanol in a 25-ml graduated
cylinder. Bring the volume up to 52 ml with glycinate and mix. After
electrophoresis, place the acid-urea gel (volume 7--- ml) in a small pan of
transparent material and add 52 ml NBT-glycinate cocktail to cover the
gel. Place the pan on an orbital shaker at room temperature for up to 1 hr
for color to develop. Wash excess glycinate from the gel with deionized
water. The reaction can be stopped by keeping the gel in 1 M sodium
borate, pH .01 4
Chromatography
Affinity Chromatography
Use of phenyl boronate immobilized to agarose (PBA; Glyco-Gel B,
Pierce Chemical Co., and Matrix PBA, Amicon Corp.) has been reported
as an affinity matrix for the purification of ferreascidin, a dopa-containing
protein from hemocytes of the tunicate Pyura stolonifera. 72 Binding to
phenylboronate would seem to be dictated by the same condition that leads
to formation of a soluble complex between o-diphenols and borate, that
is, at pH > .8 In fact, phenylboronate is considerably less predictable.
Protein must be soluble and sufficiently stable in a nonborate-containing
buffer at pH >- 5.7 to allow loading and binding to the column. This is not
fulfilled by Mefp-1 and 2- or VpB and C, which precipitate or adsorb
irreversibly at or above neutral pH. Ferreascidin is loaded and bound to
Matrix PBA-10 in 0.25 M ammonium acetate, pH 8.5. After nonbinding
proteins are washed out with the same buffer, those coupled by diols other
than o-diphenols are eluted with 1.0 M Tris with 0.2 M sorbitol and 01 mM
EDTA, pH 8.5. After a brief wash with more of the first buffer to remove
sorbitol, ferreascidin is recovered by a pH jump, i.e., eluting PBA at lower
pH, using 1 M sodium acetate, pH 3.0. PBA is more broadly applicable to
the isolation of dopa peptides. However, even with these there have been
unexpected results. After loading trypsin-digested VpB to Glyco-Gel B,
for example, Waite and Rice-Ficht 2 report on the acid elution of a mere
handful of dopa-containing peptides. More than 20 or so other dopa-con-
taining peptides do not bind. 82 Since most of the latter are acidic, nonbinding
may be a consequence of the mutual repulsion between boronate and acidic
groups on the peptides.
72 C. J. Hawkins, M. F. Lavin, D. L. Parry, and I. L. Ross, Anal, Biochem. 159, 187 (1986).
82 j. H. Waite and A. M. Rice-Ficht, Mol. Biochem. Parasitol. 54, 143 (1992).
1 GNINIATNOC-APOD SNIETORP 9
leG noitartliF
Reversed-phase HPLC is a godsend for the purification of many dopa-
containing proteins that are otherwise too adsorptive to be recoverable
from other chromatographic media. Indeed, C-8 reversed-phase HPLC is
the only chromatography necessary for aloicsaF vitelline proteins B and
C. For mussel proteins, however, a prior application to Sephadex G-200
or -150 is helpful to improve separation between Mefp-1 and -2. 4 For an
extraction from 30 g mussel feet, be prepared to fill a large column (3 x
001 cm) with Sephadex G-200 (20-30 ml/g dry gel) preswollen in an excess
volume of %5 acetic acid for at least 72 hr at room temperature. Degas
the swollen resin for at least 30 min with a vacuum aspirator. After pouring
the resin into the column, elute with at least two column volumes of %5
acetic acid at room temperature before loading the protein. Flow should
be between 1 and 3 ml/hr. Eluant is monitored at 082 nm, and Mefp-1 can
be expected to elute immediately following the void volume. The resin is
best used only once. Column fractions absorbing at 280 nm and reacting
with Arnow's method should be subjected to acid-urea polyacrylamide gel
electrophoresis. Those with protein staining with Coomassie blue R-250,
NBT, and comigrating with Mefp-1 should be pooled, frozen at -80 ,° and
lyophilized prior to HPLC.
High Performance Liquid yhpargotamorhC
slairetaM
HPLC gradient system with UV detector
C-8 analytical or preparative reversed-phase column such as Brownlee
Aquapore RP-300 (always include in-line prefilter and C-8 guard
column)
Solvent A 0.1% (v/v) trifluoroacetic acid made with Milli-Q water
that has been previously filtered through 0.45-/xm nylon-66 filter
and degassed
Solvent B (HPLC-grade acetonitrile with 0.1% trifluoroacetic acid)
Sample loading syringes (100 and 2000/zl)
Fraction collector
.erudecorP Reversed-phase HPLC has played a critical role in the puri-
fication of more than 01 different dopa-containing proteins. 92'22'91'5'4'2 Run-
ning conditions in all of these are quite similar. At the pre-HPLC stage,
dopa-containing proteins, regardless of source, are usually found in a small
volume (1-2 ml) of %5 acetic acid with or without urea. This solution
92 j. H. Waite, T. J. Housley, and M. L. Tanzer, yrtsimehcoiB 24, 5010 (1985).