Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus viridis viridis)

Pergamon Toxicon. Vol. 34. No. 4, pp. 417-434. 1996

Copyright 0 1996 Elsevier Science Ltd. All rights resewed

0041-0101(95)00154-9 Printed in Great B&din

W41-0101/96 515.W + 0.W

EVIDENCE FOR ISOMERIZATION IN MYOTOXIN

A FROM THE PRAIRIE RATTLESNAKE

(CROTALUS VIRIDIS VIRIDIS)

MICHAEL P. O’KEEFE,’ DOBRIN NEDELKOV,2 ALLAN L. BIEBER2* and RONALD A. NIEMANZ

‘Department of Chemistry, United States Military Academy, West Point, NY 10996, U.S.A. and ‘Department of Chemistry and Biochemistry, Arizona State University, Tempe, Ai! 85287,

U.S.A.

(Received 1 September 1995; accepted 23 November 1995)

M. P. O’Keefe, D. Nedelkov, A. L. Bieber and R. A. Nieman. Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus uiridis viridis). Toxicon 34, 417434, 1996.-Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25°C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5°C the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional ‘H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers;

however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5°C. Copyright 0 1996 Elsevier Science Ltd.

INTRODUCTION

The prairie rattlesnake (Crotalus viridis uiridis), a subspecies of the western rattlesnake, delivers a venom which contains many components that elicit various systemic and localized responses in victims. One component, myotoxin a, is a small 42-residue protein that induces myonecrosis upon envenomation (Mebs and Ownby, 1990; Ownby and

*Author to whom correspondence should be addressed.

417

418 M. P. O’KEEFE et al.

Colberg, 1988; Cameron and Tu, 1977). Myotoxin a was shown to inhibit and uncouple Ca*+ uptake from Ca*+-dependent ATP hydrolysis in isolated sarcoplasmic reticulum (SR) vesicles, suggesting that in situ it may bind to Ca2+-ATPase and inhibit the uptake of Ca*+, possibly leading to higher concentrations of myoplasmic Ca*+ (Volpe et al., 1986). Myotoxin a and peptides corresponding to its N- and C-fragments have been shown to bind to Ca2+-ATPase and to a 57,000 mol. wt SR protein (Utaisincharoen et al., 1991) and inhibit Ca2+ uptake by isolated SR vesicles (Baker et al., 1992). Recently, a synthetic peptide corresponding to myotoxin a residues 29-42 was shown to inhibit the activity of purified SR Ca*+-ATPase. Inhibition was decreased at low pH and at high ionic strength, suggesting important charge interactions in binding. This peptide also seemed to inhibit the binding of Ca2+ to ATPase in a pH-dependent manner (Baker et al., 1995). Myotoxin a was reported to be the most powerful known stimulator of Ca*+ release from heavy fractions of SR, inducing Ca*+ release at concentrations lower than those required for significant Ca*+-ATPase inhibition. Myotoxin a appears to affect SR Ca2+ release channels in a manner unique from other potentiators, such as caffeine, that affect ryanodine-associated Ca’+-induced Caz+ channels (Furukawa et al., 1994). ‘251-labeled myotoxin a did not bind to the purified ryanodine receptor, implying that it may bind to a unique Ca2+ release channel or to a modulator of Ca2+ release (Ohkura et al., 1995). Radiolabeled myotoxin a bound specifically to calsequestrin (55,000 mol. wt) (Ohkura et al., 1994) which may be involved in the process by which myotoxin a increases the sensitivity of Ca2+-dependent Ca*+ release (Yudkowsky et al., 1994). The potency and unique action of myotoxin a make it a highly valuable biochemical tool for studying the mechanism of Ca2+ release from the SR.

The primary structure of myotoxin a contains three proline residues and is cross-linked by three disulfide bonds (Fox et at., 1979). Myotoxin a belongs to a unique, yet highly homologous family of small, basic proteins found, thus far, only in rattlesnake venoms (Bieber et al., 1987; Aird et al., 1991). These members include myotoxins from C. durissus terrificus (Laure, 1975) C. udumunteus (Samejima et al., 1991), C. u. concolor (Bieber et al., 1987) and C. ~1. helleri (Meada et al., 1978). A complementary DNA library constructed from messenger RNA of C. durissus terrz$cus revealed multiple nucleotide sequences for myotoxin, predicting the existence of additional forms of crotamine (Smith and Schmidt, 1990). Several highly homologous myotoxins, in addition to myotoxin a, have been identified in the venom of a single specimen of C. v. viridis (Griffin and Aird, 1990; Aird et al., 1991).

Using one- and two-dimensional ‘H-NMR techniques, Henderson et al. (1987) and Murchison (Ph.D. Dissertation, Arizona State University, 1989) were somewhat successful in assigning specific proton peaks to amino acid residues in the myotoxin a sequence. However, both reported the anomaly of too many peaks in the NMR spectra for the number of protons in a single form of myotoxin a. The excess peaks led to ambiguities in interpreting the spectra and, therefore, to an incomplete set of peak assignments. The excess NMR peaks probably derive from the co-purification of similar proteins with myotoxin a. Aggregation (Henderson, Ph.D. Dissertation, Arizona State University, 1986), cis-truns isomerization at one of the three proline residues (Murchison, Ph.D. Dissertation, 1989) and other proposed disulfide arrangements (Teno et al., 1990) are possible causes of the anomalous NMR data.

Isomerization has affected the ability to analyze other small proteins by NMR. Conformational heterogeneity due to cis-truns isomerization about a predominately cis-proline peptide bond hindered the NMR structural studies of sea anemone

Myotoxin a Isomerization 419

anthopleurin-A (Scanlon and Norton, 1994). The NMR spectra of human granulocyte colony-stimulating factor showed several low intensity ‘satellite’ peaks for residues near prolines, suggesting structural heterogeneity from c&tram isomerization (Werner et al., 1994). Similar minor peaks were observed in NMR spectra of salmon calcitonin, in which the trans-isomer is dominant (Amodeo et al., 1994). Amodeo points out that when only some residues generate satellite resonances, weak nuclear Overhauser enhancement spectroscopy (NOESY) peaks from protons close to one another in the minor isomer are not distinguishable from weak peaks from protons farther apart in the major isomer. One must carefully consider the consequences of such overlapping spectra when attempting to calculate structures that satisfy the NOE constraints. One approach is to produce and study a mutant that substitutes glycine for the isomerizing proline residue. In NMR studies with such a mutant of calbindin Dsk, heterogeneity was not evident from the spectra and revealed a global folding identical to that of the major trans-isomer (Kbrdel et al., 1990).

In this study, we assign additional proton resonances in the two-dimensional ‘H-NMR spectra of myotoxin a, including the major forms of the proline peptide bonds. We demonstrate by reverse-phase high-performance liquid chromatography (RP-HPLC) and capillary zone electrophoresis (CZE) that heterogeneity is a major problem. We characterize this heterogeneity at different temperatures and pH values and use matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry to show that resolved peaks contain proteins with indistinguishable masses.

MATERIALS AND METHODS

Myotoxin a purification Myotoxin a was purified from Crotnlus ciridis oiridis venom (Laser Lab, Salt Lake City, U.S.A.) using

essentially the method of Henderson (Ph.D. Dissertation, Arizona State University, 1986). One gram of dry crude venom was dissolved in 5 ml of 0.1 M KCljO.05 M Tris buffer (pH 9.0) and centrifuged 5 min at 200 x g at 4C. The pellet was resuspended in 5 ml of the same buffer and the suspension was again centrifuged under the same conditions, The collected supernatant solutions from both centrifugations were pooled. This sample was run on to a Fractogel TSK CM650S (EM Sciences, Gibbstown, NJ) carboxymethyl cellulose cation-exchange column (2.5 x 37.5 cm; Pharmacia) at 2°C in 0. I M KCljO.05 M Tris buffer (pH 9.0) at a flow rate of 1.5 ml/min and was followed by a linear salt gradient from 0.1 to 1 M KCl. Absorbance at 280 nm was measured on an Instrumentation Specialties Co. (ISCO, Lincoln, NE) type 6 optical unit, amplified by an ISCO Model UA-5 absorbance/fluorescence monitor. Fractions were collected and those corresponding to the second major peak were pooled. Each sample was ultrafiltered in a 43 mm Amicon model 52 concentrator using a Diaflo YM2 membrane filter (Amicon, Beverly, MA) at 2’C with 55 psi N*(g) through several dilution/concentration cycles. Concentrates were lyophilized and kept desiccated at -20°C until used.

NMR sample preparation Lyophihzed myotoxin a was dissolved to c. 34 mM in 85% H20/15% DsO (Aldrich Chemical Co., Milwaukee,

WI) with a small amount of 3-(trimethyl)propionic-2,2,3,3,-d4 acid (TSP) (Aldrich Chemical Co.) to a total volume of c. 0.8 ml. The pH was measured with a Lazar PHR-146 micro combination electrode and adjusted to 3.5 with I M and 0.1 M HCI. A 50 ~1 aliquot was diluted into 950 ~1 H10, and absorbance of the final solution was measured at 280 nm with a Varian DMS 100s UV/Visible spectrophotometer. An extinction coefficient of ~280 = 2.27 rngm ’ mlcmm ’ (Allen et al., 1986) was employed to calculate the concentration of the sample.

NMR spectra iH-NMR spectra were acquired on a Varian Unity 500 spectrometer controlled by a Sun 41260 workstation

running Varian VNMR software. The spectrometer was operated at 499.843 MHz with a sweep width of 6199.6 Hz at 25°C. Double quantum filtered correlated spectroscopy (DQFCOSY) (Rance et al., 1983) experiments were normally acquired with 4 K points, 48 scans, and 800 increments. Total correlation spectroscopy (TOCSY) (Bax and Davis, 1985) spectra were normally acquired with 2 K points, 32 scans, and 600 increments with mixing

M. P. G’KEEFE et al. 420

times from 20 to 100 msec. NOESY (Kumar et al., 1980) experiments were usually acquired with 2 K points, 96 scans. and 512 increments with mixing times from 50 to 350 msec. Spectra were acquired with phase-sensitive detection using the hypcrcomplex method (States et al., 1982). Spectra were analyzed on a Silicon Graphics Indigo R3000 workstation using Felix 2.05 (Hare Research, Bothell, WA). The spectra were Fourier transformed with a sine square windowing function and zero filled to a typical digital resolution of 3.02 Hz/point in each dimension. The resultant 2 K x 2 K real matrices were inspected and a suitable data cut-off threshold was set. The 20 plots of data in these matrices were then used to make the spectral assignments following the general strategy of Wiithrich (1986).

Reverse-phase HPLC Analytical RP-HPLC separations were performed on a Zorbax SBX3 (4.6 mm ID x 15 cm) column

(Rockland Technologies) connected to a System Gold programmable solvent module 125 and detector module 166 (Beckman Instruments, Fullerton, CA). A volume of 25 ~1 of 0.2 mM myotoxin a in 20% acetonitrile (JT Baker, Phillipsburg, NJ) was injected. The column was eluted with a linear gradient of 20 to 21% acetonitrile in 0.01 M aqueous trifluoroacetic acid (Sigma Chemical Co., St Louis, U.S.A.) at a flow rate of 1.0 mI/min over 15 min. Absorbance at 220 nm was measured, and the resulting chromatograms were analyzed with System Gold software (Beckman Instruments). The fractions were collected manually and incubated at room temperature or on ice before injection. The injection volume for each fraction was 50 ~1. The HPLC separations were either performed at room temperature (c. 25°C) or chilled to c. 5°C (9°C or less was measured at the outlet of the detector), with the column, sample loop, and solvents in ice baths.

Semi-preparative RP-HPLC was performed on a Waters 600 E system controller, a Waters 717 autosampler, and a Waters 996 photodiode array detector using Millennium 2010 Chromatography Manager v.2.00 software (Millipore Corp., Bedford, MA). The Zorbax 3OOSBC3 (9.4 mm ID x 25 cm) column was injected with a volume of 50 ~1 or 100 ~1 of myotoxin a (0.0625-2 mg/ml) and eluted with a typical gradient of 17-23% acetonitrile in 0.01 M aqueous trifluoroacetic acid over 20 min at a flow rate of 4 ml/min. Low temperature separations were performed by maintaining the solvents in an ice bath. Temperatures of 25°C and higher were controlled by both the autosampler (tray compartment) and the system controller (column heater). The autosampler, however, had a maximum setting of 40°C. The column heater was run up to 80°C but since the solvents were at room temperature prior to entering the system, the actual temperatures of the separations were probably lower than the temperature settings which are reported here. Absorbance was measured at 220 nm and 280 nm, and the resulting chromatograms were analyzed with the Millenium software.

Capillary zone electrophoresis CZE separations were performed on a P/ACE System 2050 using an eCAP Amine Capillary (50 pm ID x 47

cm), both from Beckman Instruments. This capillary employs a polyamine modified surface that creates a strong cationic charge on the capillary wall, minimizing the interaction with the highly basic myotoxin a. The system was operated in reverse-polarity mode (cathode at the inlet; anode at the outlet), and the direction of electro-osmotic flow (EOF) is reversed from that in an untreated fused silica capillary. Myotoxin a, although positively charged, moved toward the anode because of the overwhelming EOF. All CZE separations were performed at 25°C and a constant potential of 20 kV. The 0.3 mM or 1.0 mM myotoxin a samples were injected under nressure for 5 or 30 sec. All samoles had less than 0.1% of dimethvl formamide (DMF) to mark the EOF. The running buffers were 0.1 M acetate/phosphate (pH 4.0, 4.5, 5.0, 5.5jand 0.1 M phosphate (pH 6.0, 6.5, 7.0, 7.5). Detection was by UV absorbance at 214 nm. Fraction collection was performed as described by Biehler and Schwartz (1991).

Mass spectrometry A LaserTec Research MALDI time-of-flight mass spectrometer (PerSeptive Biosystems, Cambridge, MA) was

used to assess the masses of components within the collected RP-HPLC and CZE fractions. The matrix used was a-cyano-4-hydroxycinnamic acid, and samples were prepared as described by Beavis et al. (1992).

RESULTS

NA4R spectra The fingerprint (NH-C”H) region of the DQFCOSY spectra should have a maximum

of 46 NH-C”H peaks if the C”H proton peaks of all five glycine residues were distinguishable and if the peaks of the N-terminal protons were visible. At the lowest


threshold of display, 57 peaks appear clearly in this region (Fig. 1). Many instances of weak peaks appearing very close to, or partially overlapping, strong peaks are evident. Peaks were readily picked and correlated, except in the far upfield region (0.5-3.5 ppm) where overlap became severe. The TOCSY spectra most useful for assigning spin systems came from a mixing time of 60 msec, though all the spectra suffered to some extent from distorted peaks which were probably caused by slightly offset overlapping peaks (results not shown). The NOESY spectra could be effectively used with the DQFCOSY spectra in the fingerprint region to connect the NH and C”H peaks of adjacent residues. Proline residues were identified by their unique spin systems. Pro-13 and Pro-21 were specifically assigned through NOESY Pro-C”H,-NH 1 + , cross-peaks to Lys-14 and Ser-22, respectively. Pro-20 was assigned as the remaining proline spin system. Specific assignments were made from the resulting contiguous chains of spin systems (Fig. 2). A nearly complete listing of assignments appears in Table 1. Of particular note, a strong NOESY cross-peak was observed between C’H of Ile-19 and C”H of Pro-20 (Fig. 3) uniquely indicative of a

q

- 4.5

Fl

ppm

I I I I I I 6.0 I.5 10.0 9.5 9.0 8.5 8.0 7.5

F2 mm Fig. 1. Fingerprint region (NH-C”H) of DQFCOSY of myotoxin a.

Myotoxin Q was prepared to a concentration of 3.4 mM in 85% HsO/lS% DIO with TSP, pH 3.5. DQFCOSY 500 MHz ‘H-NMR spectra were acquired at 25°C. Peaks are labeled with their residue assignments for the major form of myotoxin 4. Unlabeled peaks from minor components are present in the sample. Note that many minor peaks appear in the vicinity of stronger, identified

peaks (e.g. Cll, F12, W34, and K38).


I ,i

,J , ,,,I

.5 10.0 9.5 9.0 8.5 8.0 7.5

4.5

Fl pm

5.5

8.0

F2 wm Fig. 2. Fingerprint region (NH-C”H) of NOESY of myotoxin a.

Myotoxin a was prepared to a concentration of 3.4 mM in 85% H,O/15% D20 with TSP, pH 3.5. NOESY 500 MHz ‘H-NMR spectra were acquired at 25°C. Intraresidue NH-C”H peaks are labeled with their residue assignments for the major form of myotoxin a; sequential interresidue NH-C’H peaks are not labeled. Sequential connections are shown for Lys-7 to Phe-12 (- -), Pro-13 to Ile-19 (- - -), and Pro-21 to Ser-41 (F ). The peaks with labels in parentheses are visible in spectra at 50°C. Note the appearance of minor peaks in the vicinity of peaks assigned to the major form

of myotoxin a (e.g. the Cys-11 C”H to Phe-12 NH peak).

cis-proline peptide bond (Wiithrich, 1986). No such peak was observed for the other two proline residues. A strong NOESY cross-peak was observed between C”H of Pro-20 and C6H of Pro-21, confirming the presence of a trans-proline peptide bond between these two residues (result not shown).

RP-HPLC and MALDI mass spectrometry analyses The BP-HPLC analysis of the purified myotoxin a at c. 25°C resulted in a separation

of two peaks, labeled fractions A and B, which elute in approximately a 1:4 molar ratio based on peak areas (Fig. 4a). When subjected to MALDI mass spectrometry, each fraction yielded a primary mass of 4830 mol. wt (Fig. 4f, g), corresponding to an uncorrected mass (no internal mass standard) for myotoxin a, calculated at 4824 mol. wt (Fox et al., 1979). Each fraction, after being incubated at c. 25°C for 30 min (fraction A)

Tab

le

1.

‘H-N

MR

ch

emic

al

shif

ts

of

myo

toxi

n a

at

pH

3.5,

25

°C

(*5O

”C)

(ppm

)

Res

idue

N

H

C”H

C

BH

C

H

CSH

O

ther

Tyr

-1

Lys

-2

Gln

-3

cys-

4 H

is-5

L

ys-6

L

ys-7

G

ly-8

G

ly-9

H

is-1

0 cy

s-11

Ph

e-12

Pr

o-

13

Lys

-14

Glu

-15

Lys

- 16

Il

e- 1

7 C

ys-1

8 Il

e-19

Pr

o-20

Pr

o-2

I Se

r-22

Se

r-2

Asp

-24

Leu

-25

Gly

-26

Lys

-21

Met

-28

Asp

-29

cys-

30

Arg

-3

1 T

ip-3

2 L

ys-3

3 T

rp-3

4 L

ys-3

5 C

ys-3

6 cy

s-37

L

ys-3

8 L

ys-3

9 G

ly-4

0 Se

r4

1 G

ly-4

2

7.67

8.

24

8.97

8.

63

8.99

7.

36

7.89

8.

03

8.34

8.

73

10.4

4

8.05

8.

60

7.69

8.

33

8.82

8.

09

7.91

8.

50

7.49

8.

88

8.63

8.

54

8.69

8.

77

8.48

8.

07

8.11

8.

45

8.94

9.

89

9.02

9.

17

9.33

8.

45

8.64

8.

31

4.10

3.

94

4.52

4.

96

4.48

4.

35

4.19

4.

88

4.62

5.

71

4.97

3.

66

3.66

3.

97

3.98

3.

98

4.43

4.

18

5.01

4.

44

4.27

4.

29

5.06

4.

45

4.33

4.

00

3.93

4.

74’

4.47

4.

11

4.48

3.

5 5.

11

4.76

’ 5.

20

5.88

4.

39

4.43

4.

14

4.52

3.16

2.

98

1.39

1.

76

3.78

3.

38

3.03

3.

14

2.99

1.

75

1.97

1.

67

1.74

3.

11

1.71

2.

54

2.51

4.

03

4.11

3.

26

1.84

3.

89

1.92

2.

23

3.15

1.

57

3.32

2.

13

3.47

3.24

3.

27

I .95

1.

91

4.04

3.

91

2.69

2.

51

2.89

2.

96

2.46

1.

50

1.88

2.83

2.37

2.

01

3.92

3.

92

2.39

1.

45

1.79

3.08

1.

32

3.25

3.07

2.86

3.

11

1.75

3.83

1.32

1.

16

2.31

2.

19

0.85

0.

77

1.99

1.

80

2.23

2.

14

1.55

1.

34

2.91

3.63

3.

87

2.55

3.52

7.05

(2,6

8)

6.80

(3,5

8)

1.63

’ 1.

15’

1.75

’ 0.

85’

I .88

+ 2.

06’

1.54

’

8.58

(2H

) 6.

99(4

H)

7.44

(3,

5H)

7.37

(48)

7.

28(2

,6H

)

1.36

’ 1.

11’

0.91

’ 1.

15’

0.80

’ 5 0

1.75

’ 0.

84’

2.51

’ 2.

40’

1.86

’

2.49

6.

62(N

t)

10.2

8(N

t)

7.64

(48)

7.

56(7

8)

7.37

(2H

) 7.

30(6

H)

7.18

(5H

) 1.

41’

0.79

’ 0.

58’

10.2

7(N

t)

7.54

(7H

) 7.

39(2

8)

7.26

(6H

) 7.

07(5

H)

1.62

’ 1.

27’

1.54

’ I .

46*

3.01

’ 1.

72’

1.53

’

Myo

toxi

n a

was

pre

pare

d to

a c

once

ntra

tion

of 3

.4 m

M

in 8

5%

Hz0

/15%

D

lO

with

T

SP,

pH

3.5.

500

MH

z ‘H

-NM

R

spec

tra

wer

e ac

quir

ed

at 2

5°C

an

d 50

°C.

Che

mic

al

G

shif

ts

are

repo

rted

fo

r 25

°C

exce

pt

for

thos

e in

dica

ted

(*)

at

WC

, w

hich

w

ere

not

visi

ble

at

25°C

du

e to

ov

erla

p w

ith

the

wat

er

reso

nanc

e.

The

ch

emic

al

shif

t va

lues

la

bele

d (‘

) co

uld

not

be

assi

gned

to

sp

ecif

ic

prot

on(s

) in

th

e re

spec

tive

amin

o ac

id

due

to

a se

vere

ov

erla

p of

th

e pe

aks.


4.2

F2 w*

Fig. 3. Ile-19 C”H to Pro-20 C”H cross-peak in NOESY of myotoxin a. Myotoxin a was prepared to a concentration of 3.4 mM in 85% HzO/lS% D20 with TSP, pH 3.5. NOESY 500 MHz ‘H-NMR spectra were acquired at 25°C. A strong NOESY cross-peak is observed between C”H of Ile-19 and C”H of Pro-20, uniquely indicative of a cis-proline peptide

bond (Wiithrich, 1986).

or 1 hr (fraction B), was injected into the column and yielded two peaks which eluted in positions corresponding to the two original fractions (Fig. 4b, d). After 24 hr of incubation at c. 25°C injection of each original fraction A and B yielded the same elution profile (Fig. 4c, e) as that from the original sample of myotoxin a (Fig. 4a).

The same set of RP-HPLC separations was repeated with the samples incubated on ice and with the column, loop, and solvents chilled by ice. Temperatures of 9°C or lower were observed at the detector outlet. The results of these cold separations reveal that the interconversion of fractions is very slow, so long as they are kept at ice-bath temperatures (Fig. 5a-g). Even after 4 days, the original peak area ratio of 1:4 was not reached by either fraction A or B. Lower peak heights can be seen in the 24 hr injections for both fraction A and B (Fig. 5c, f) than in the 30 min and 1 hr injections (Fig. 5b, e). The lower peak height is obvious in Fig. 5c and occurs in both peaks (eluting at 3.2 and 4.1 min), suggesting that it is not due to an interconversion of fractions. The reason for this occurrence is difficult to understand. The experiments were done in triplicate and the same result was observed. A possible explanation is that myotoxin a, a highly basic protein, may be binding to the surface of the glass or polypropylene vials used to incubate the fractions, as suggested by Baker et al. (1993). Nevertheless, the interconversion at later times was as


expected (compare the peak ratios in Fig. 5c, d) although at a very slow rate owing to the low temperature of separation. In addition to showing the temperature dependence of fraction interconversion, the results show that fraction A converted to fraction B more quickly than fraction B converted to fraction A.

1.0

0.6

Fraction B

0.6 Fraction A

Ei

2 0.4

0.2

0.0

\

J --

0.030

0.025

0.020

a b

0.005

0.000

-0 005

i

I I I I I I

0 6 7 0 1 2 3 4 5 6 7 1 2 3 4 5

Time [min] Time [min]

0.10

0.06

0.06

z

2 0.04

0.030 T---- 0.025

l- d C

0.020

0.015 0

2 0.010

-0.065 1 I I I I ! , 0 1 2 3 4 5 6

Time [min]

1 7 0 1 2 3 4 5 6 7

Time [min]

Fig. 4 (a)-(d) (caption ooerieuf).

426

0.10

0.06

0

z a 0.04

M. P. O’KEEFE et al.

e

1

1 2 3 4 5 6 7

Time [min] 2000 4000

m/z

4000

m/z

Fig. 4 W(g)

6(

Fig. 4. Room-temperature RP-HPLC separation of myotoxin a, injection of the separated fractions, and mass spectrometry analyses.

The RP-HPLC runs were performed on a Zorbax SB-C3 (4.6 mm ID x 15 cm) column eluted with a linear gradient of 20-21% acetonitrile (ACN) in 0.01 M aqueous trifluoroacetic acid (TFA) over IS min at a flow rate of 1.0 ml/min. A 25 ~1 sample of myotoxin a (0.2 mM in 20% ACN) was applied to the column (a). The eluted fractions A and B were collected manually, incubated at room temperature (c. 25°C) and injected into the column (V,tiw = 50 ~1). The injections were made after 30 min (b) and c. 24 hr (c) for fraction A and after 1 hr (d) and c. 24 hr (e) for fraction

B. The mass analyses of fractions A and B are shown in (f) and (g), respectively.


When myotoxin a was separated by semi-preparative RP-HPLC over a range of temperature settings from 25°C to 65°C (the actual temperatures being somewhat lower

at the high end of the range - see Materials and Methods), the resolution of peaks corresponding to fractions A and B decreased until they merged (Fig. 6). The overall

7------ a

0.8

1

0.6 -

0

ti a 0.4 -

0.2 -

0.0 _J V

0.030

0.025

0.020

0.015

Ei

2 0.010

0.005

I I I I I 1 I

0 1 2 3 4 5 6 7

Time [min]

I C

0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7


0.030

0.025

0.020

0.015 0

z a 0.010

0.005

0.000

-0.005

0.030

0.025

0.020

0.015

i?i

0.010

0.005

0.000

-0.005

4

b

I I I / I I

0 1 2 3 4 5 6 7

Time [min]

d

Fig. 5. (a)-(d) (caption ooerleuf)

428

0.08

0.06

z

T

0.04

0.02

0.00

M. P. O’KEEFE er al.

e

:

/ I I I I I

0.08 -

0.06 -

E (v 0.04

a r'

0.02

I

f

1 0 12 3 4 5 6 7 0 12 3 4 5 6 7


0.08

0.06

Ei (\1 0.04

a

0.02

0.00

r g

I I I I I I I -1 0 12 3 4 5 6 7

Time [min]

Fig. 5 (e)-(g)

Fig. 5. Cold-temperature RP-HPLC separation of myotoxin a and injection of the separated fractions.

The column, gradient, sample concentration and injection volume for each run was as described in Fig. 4. The solvents, column, and sample loop were kept on ice. The sample of myotoxin a was incubated on ice prior to injection. After the separation (a), the eluted fractions A and B were directly injected into the column using an ice-cold syringe. The injections were made after 30 min (b), c. 24 hr (c) and c. 96 hr (d) for fraction A, and after 1 hr (e), c. 24 hr (f) and c. 96 hr (g) for

fraction B. Between injections, both fractions were kept in an ice bath.


reduction in retention times with increasing temperature is indicative of the reduced hydrophobic interactions. The apparent rate of interconversion of fractions A and B increased significantly with temperature.

Samples of myotoxin a over a range of concentrations from 0.0625 mg/ml to 2 mg/ml (0.013 mM to 0.41 mM) were separated at 25°C by semi-preparative RP-HPLC. The peak areas corresponding to fractions A and B did not vary significantly from the c. 1:4 ratio seen in Fig. 4a over the entire range of concentrations (results not shown).

CZE separations CZE analyses of myotoxin a samples were performed using an amine-coated column,

A 0.3 mM sample of myotoxin a was subjected to CZE analysis with running buffers at

0.41

0.21

0.01

AZ:

0;

oi

Oi-

10

Fig. 6. RP-HPLC chromatograms of myotoxin a at various temperatures. A 50 ~1 sample of myotoxin a (0.2 mM) was separated on a semi-preparative C3 column with a gradient of 17-23% ACN with 0.01 M aqueous TFA over 20 min at a flow rate of 4 ml/min. Temperatures were controlled within the sample tray compartment up to 40°C. Temperatures above 40°C represent only the column heater setting. Solvents were at room temperature prior to entering the system, so the actual temperatures of the separations were likely less than those values

shown Absorbance was measured at 220 nm.


pH=7.5

DMF

pH=4.0

DMF

I I I I I I

6 6 10 12 14 16

Time [min] Fig. 7. Capillary zone electrophoresis separation of myotoxin a at different pH values.

All runs were performed on an eCAP amine coated capillary (50 pm ID x 47 cm) from Beckman Instruments, Inc. The separations were performed at 25°C and a constant voltage of 20 kV. The running buffers were 0.1 M acetate/phosphate, pH 4.0 and 0.1 M phosphate, pH 7.5. A sample of 0.3 mM myotoxin a was injected under pressure for 5 set in both runs. The sample of myotoxin

a contained less than 0.1% dimethyl formamide (DMF), which served as an EOF marker.

different pH values (see Materials and Methods). Each run resulted in separation of myotoxin a into the two characteristic peaks. A typical electropherogram is shown in Fig. 7. The use of phosphate in all the buffers was critical to obtain good separation of peaks. Phosphate, as a polyprotic anionic buffer, is used to reduce the electroosmotic flow, thereby improving the resolution. The higher concentration of phosphate in the pH 7.5 buffer resulted in a much slower EOF (Fig. 7). As a result, the migration times for myotoxin a at pH 4.0 and 7.5 appear to be almost identical, even though the electrophoretic velocity of myotoxin a at pH 4.0 is higher than at pH 7.5. In order to get enough sample for further analyses, a higher sample concentration (1 mM) was employed (Fig. 8a). Owing to mass overloading, the peaks show a tailing profile. Each peak was assayed by MALDI mass spectrometry and the primary mass of each was consistent with myotoxin a (results not shown). The injection of fraction B from the separation of myotoxin (1 shown in Fig. 8a after 4 hr at c. 25°C (followed by 2 days of storage at c. -20°C) resulted in a separation into two peaks with a ratio of areas of c. 1:4 (Fig. 8b). The results of CZE separations are consistent with the results of RP-HPLC separations; in each case, myotoxin a separated into two fractions that could interconvert.

DISCUSSION

Analysis of NMR data from myotoxin a is hindered by the presence of more than one form of the protein, leading to many weak peaks that partially overlap strong peaks (Fig. 1). The strong COSY and TOCSY peaks associated with the major form of myotoxin

Myotoxin rr lsomerization 431

a and the connecting NOESY peaks led to the assignment of the vast majority of proton resonances with a great deal of confidence (Table 1). However, the assembly of a definitive set of NOE distance constraints for calculating a three-dimensional structure is greatly hindered by ambiguities. Weak NOE peaks may result from small distances between interacting protons in the minor forms or from protons separated by greater distances in the major form. Pro-20 shows a strong C”H-C”H cross-peak with Ile-19, clearly indicating that the cis-configuration is the predominant form for the peptide bond between these two residues. The identification of this cis-proline peptide bond suggests that cis-tram isomers may be responsible for some of the sample heterogeneity.

The same primary masses for fractions A and B from 25°C RP-HPLC (Fig. 4f, g) suggest that the two fractions contain conformational isomers of myotoxin a, which in solution can be separated by RP-HPLC based on their difference in hydrophobicity. Each isolated fraction produced both fractions in the original molar ratio when injected 24 hr later at c. 25°C (Fig. 4c, e). Thus, the isomers must convert slowly on the HPLC time scale, but achieve equilibrium within 24 hr at that temperature. When this procedure was performed at c. 5”C, the interconversion was greatly slowed, so long as the collected fractions remained in an ice bath. Even after 4 days the equilibrium ratio of the original sample was not reached by either fraction (Fig. 5). In contrast, when subjected to elevated temperatures, the interconversion becomes rapid on the HPLC time scale and peaks A and B merge to a single peak (Fig. 6). C&tram prolyl peptidyl isomers of dipeptides have been effectively separated by RP-HPLC (Wutte et al., 1994) and it is not unreasonable to

0.06

0.07

0.06

0.05

Tf 0.04

r; a o.03

0.02

0.01

0.00

-0.01

Fraction B

\

Fraction A

\ DMF

/

a c I I I I I I

0 5 10 15 20 25 30

Time [min]

0.005

0.004

r 0.003

1

0.002 w

;;1

a o.ool

0.000

-0.001

_.

I

-0.002

DMF \

0 5 10 15 20 25

Time [min]

separation of Fraction B 1

Fig. 8. Capillary zone electrophoresis separation of myotoxin a and injection of the separated fraction.

The column, temperature, and voltage were as described in Fig. 7. A 1 .O mM sample of myotoxin a containing less than 0.1% DMF was injected under pressure for 5 set (a) and fraction A was collected in the outlet vial. Fraction B was eked under pressure into a separate vial containing 20 ~1 HzO. Fraction B was injected under pressure for 30 set (b). Absorbance was measured

at 214 nm.


suggest that RP-HPLC may be effectively separating cis-tram isomers of myotoxin a. Other closely related myotoxins, which vary slightly in sequence from myotoxin a (Griffin and Aird, 1990; Aird et al., 1991), may be present to a minor extent (note the slight shoulder on the higher m/z side of the myotoxin a peak in Fig. 4f, g). However, these are clearly not the main components of the two interconverting fractions (A and B) observed by RP-HPLC.

Comparable results were obtained from CZE, namely, separation of myotoxin a in two fractions with the same primary mass and interconversion of the second fraction to yield both fractions in the original molar ratio (Fig. 8a, b). However, one must bear in mind that two quite different techniques were used to generate the results. Whereas separation of species in RP-HPLC is based on difference in hydrophobicity, the charge to mass ratio plays a crucial role in CZE. Since both fractions were shown to have the same mass, it is obvious that the separations in the CZE runs resulted from differences in net charge. If the separated fractions are c&tram isomers of myotoxin a, the isomerization about the proline petide bond could result in altered distribution of surface charge on the protein. While literature is available that reports separation of protein isoforms by CZE (Markland et al., 1993; Richards, 1994), this would be the first report on separation of cis-trans protein isomers by capillary zone electrophoresis.

The presence of separate RP-HPLC peaks with components of the same mass at pH 4, but not at pH 5.5, is not likely to result from a change in the protonation state of glutamate residues, as suggested by others (Griffin and Aird, 1990; Aird et al., 1991). The CZE results showed consistent separation of peaks for myotoxin a from pH 4.0 to 7.5 (Fig. 7) which clearly indicates that the presence of two forms of myotoxin a is not an artifact of the low pH conditions used for RP-HPLC separations and for NMR acquisitions. Furthermore, the results do not provide support for the idea that the two forms of myotoxin a differ by the protonation and deprotonation of glutamate residues.

Henderson (Ph.D. Dissertation, 1986) suggested that myotoxin a may aggregate in solution. This would certainly generate a form of myotoxin with different hydrophobicity than the monomer. Separation of monomers from multimers by RP-HPLC would be expected, but MALDI mass spectrometry would not distinguish between them. Since such an aggregation would be likely to occur through noncovalent intermolecular forces, equilibrium between aggregates and the monomer in solution at room temperature would seem reasonable. The simplest model would be the formation of a noncovalent dimer (D) in equilibrium with two monomers (M). This would give rise to an equilibrium constant approximately equal to the concentration of the dimer divided by the concentration of the monomer squared: K, = [D]/[M]*. If the resolved peaks represent monomeric and dimeric forms, the ratio of RP-HPLC peak areas for the two forms of myotoxin a would change in a concentration-dependent manner. However, RP-HPLC separations of myotoxin a over a 32-fold range of concentrations did not vary significantly from the c. 1:4 ratio for peaks A and B seen in Fig. 4a. Therefore, aggregation does not appear to be the source of the observed forms of myotoxin a.

The addition of cyclophilin (peptidyl prolyl cis-trans isomerase) (Harding, 1991) to fractions collected from semi-preparative ice-cold RP-HPLC separations did not increase the rate of equilibration of the isolated fractions, even when raised to an optimum pH (c. pH 8) (Harrison and Stein, 1990) and 25°C (results not shown). The isomerase activity of cyclophilin on the small molecule succinyl-Ala-Ala-Pro-Phe-p-nitroanilide has been observed directly by one-dimensional ‘H-NMR spectroscopy (Hsu et al., 1990) and through indirect spectrophotometric assays (Harrison and Stein, 1990). The catalytic


effects of cyclophilin on unfolded proteins have been observed by studying the folding kinetics of human carbonic anhydrase II (Kern et al., 1995) and RNase Tl (Schonbrunner and Schmid, 1992), for example. The inability of cyclophilin to catalyze the conversion of the isolated forms of myotoxin a does not discount the possibility that they are cis-tram peptidyl prolyl isomers, since folded myotoxin a may not be a suitable substrate for the cyclophilin.

The results of our experiments from a number of different techniques provide strong evidence for the existence of an equilibrium mixture of two isomers in solutions of myotoxin a. The exact nature of the isomers is unknown; however, the presence of the cis-Pro 20 in the major form suggests that the interconverting forms observed may be c&tram isomers. The results do not support other suggested possibilities such as aggregation or protonation. It is inappropriate at this time to speculate about effects of isomerization on the function of myotoxin a in situ. However, a better understanding of myotoxin a structure will be essential for understanding the function of myotoxin u as it gains importance as a biochemical probe of Ca*+ release in muscle cells.

Acknowledgements-This research was supported in part by NIH grant 2 R01 GM34925 and the Army Research Office, West Point, NY.

REFERENCES

Aird, S. D., Kruggel, W. G. and Kaiser, I. I. (1991) Multiple myotoxin sequences from the venom of a single prairie rattlesnake (Crotalus viridis viridis). Toxicon 49, 265-268.

Allen, H. R., Tucker, R. K. and Geren, C. R. (1986) Potentiation of the toxicity of basic peptides from rattlesnake venoms by sodium acetate. Toxicon 24, 553-558.

Amodeo, P., Morelli, M. A. C. and Motta, A. (1994) Multiple conformations and proline c&tram isomerization in salmon calcitonin: a combined nuclear magnetic resonance, distance geometry, and molecular mechanics study. Biochemistry 33, 1075410762.

Baker, B., Utaisincharoen, P. and Tu, A. T. (1992) Structure-function relationship of myotoxin a using peptide fragments. Arch. Biochem. Biophys. 298, 325-33 1.

Baker, B., Tu, A. T. and Middlebrook, J. L. (1993) Binding of myotoxin a to cultured muscle cells. Toxicon 31, 271-284.

Baker, K. J., East, M. J. and Lee, A. G. (1995) Mechanism of inhibition of Ca l+-ATPase by myotoxin a. Biochem. J. 307, 571-579.

Bax, A. and Davis, D. G. (1985) MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy. J. Magn. Reson. 65, 355-360.

Beavis, R. C., Chaudhary, T. and Chait, B. T. (1992) cc-Cyano-4-hydroxycinnamic acid as a matrix for matrix-assisted laser desorption mass spectrometry. Org. Mass Spectrom. 27, 156158.

Bieber, A. L., McParland, R. H. and Becker, R. R. (1987) Amino acid sequences of myotoxins from Crotalus uiridis concolor venom. Toxicon 25, 677-680.

Biehler, R. and Schwartz, H. E. (1991) Fraction collection of peptides using P/ACE system 2000 followed by microsequencing. In: Technical Bulletin TIBC-105, pp. l-5. Palo Alto, CA: Beckman Instruments.

Cameron, D. L. and Tu, A. T. (1977) Characterization of myotoxin a from the venom of prairie rattlesnake (Croralus viridis viridis). Biochemistry 16, 25462553.

Fox, J. W., Elzinga, M. and Tu, A. T. (1979) Amino acid sequence and disulfide bond arrangement of myotoxin a isolated from the venom of prairie rattlesnake (Crotnlus viridis viridis). Biochemistry 18, 678-684.

Furukawa, K. I., Funayama, K., Ohkura, M., Oshima, Y., Tu, A. T. and Ohizumi, Y. (1994) Ca2+ release induced bv myotoxin a, a radio-labellable nrobe havine novel Ca2+ release nrouerties in sarconlasmic reticulum. Br. J. Pharmac. 113, 233-239. _

. L

Griffin, P. R. and Aird, S. D. (1990) A new small myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis). FEBS Lert. 274, 4347.

Harding, M. W. (1991) Structural and functional features of the peptidyl prolyl cis-tram isomerase, cyclophilin. Pharmacotherapy 11, 142S-148s.

Harrison, R. K. and Stein, R. L. (1990) Mechanistic studies of peptidyl prolyl cis-tram isomerase: evidence for catalysis by distortion. Biochemistry 29, 1684-1689.

Henderson, J. T., Nieman, R. A. and Bieber, A. L. (1987) Assignment of the aromatic ‘H-NMR resonances of myotoxin a isolated from the venom of Crotalus viridis viridis. Biochim. biophvs. Acra 914, 152-161.


Hsu, V. L., Handschumacher, R. E. and Armitage, I. M. (1990) Peptidyl-prolyl c&tram isomerase activity of cyclophilin studied by one-dimensional IH nuclear magnetic resonance spectroscopy. J. Am. them. Sot. 112, 61456747.

Kern, G., Kern, D., S&mid, F. X. and Fischer, G. (1995) A kinetic analysis of the folding of human carbonic anhydrase II and its catalysis by cyclophilin. J. biol. Chem. 270, 740-745.

Kiirdel, J., For&, S., Drakenberg, T. and Chazin, W. J. (1990) The rate and structural consequences of proline cis-tram isomerization in calbindin Dyk: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant. Biochemistry 29, 4400-4409.

Kumar, A., Ernst, R. R. and Wiithrich, K. (1980) A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem. biophys. Res. Commun. 95, l-6.

Laure, C. J. (1975) Die primarstruktur des crotamins. Hoppe-Seyler’s Z. physiol. Chem. 356, 213-215. Markland, F. S., Morris, S., Deschamps, J. R. and Ward, K. B. (1993) Resolution of isofomrs of natural and

recombinant fibrinolytic snake venom enzyme using high performance capillary electrophoresis. J. Liq. Chromatogr. 16, 21892201.

Meada, N., Tamiya, N., Pattabhiraman, T. R. and Russell, F. E. (1978) Some chemical properties of the venom of the rattlesnake, Crotalus viridis helleri. Toxicon 16, 431441.

Mebs, D. and Ownby, C. L. (1990) Myotoxic components of snake venoms: their biochemical and biological activities. Pharmac. Ther. 48, 223-236.

Ohkura, M., Furukawa, K. I., Tu, A. T. and Ohizumi, Y. (1994) Calsequestrin is a major binding protein of myotoxin a and an endogenious Ca*+ releaser in sarcoplasmic reticulum. Eur. J. Pharm. 268, Rl-R2.

Ohkura, M., Furukawa, K. I., Oikawa, K. and Ohizumi, Y. (1995) The properties of specific binding site of 1*51-radioiodinated myotoxin a, a novel Ca*+ releasing agent, in skeletal muscle sarcoplasmic reticulum. J. Pharmac. exp. Ther. 273, 934939.

Ownby, C. L. and Colberg, T. R. (1988) Classification of myonecrosis induced by snake venoms: venoms from the prairie rattlesnake (Croralus viridis viridis), western diamondback rattlesnake (Crotalus atrox) and the Indian cobra (Naja naja naja). Toxicon 26, 459474.

Rance, M., Sorenson, 0. W., Bodenhausen, G., Wagner, G., Ernst, R. R. and Wilthrich, K. (1983) Improved spectral resolution in COSY ‘H NMR spectra of proteins via double quantum filtering. Biochem. biophys. Res. Commun. 117, 419-485.

Richards, M. P. (1994) Application of a polyamine-coated capillary to the separation of metallothionein isoforms by capillary zone electrophoresis. J. Chromatogr. B. 657, 345-355.

Samejima, Y., Aoki, Y. and Mebs, D. (1991) Amino acid sequence of a myotoxin from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). Toxicon 29, 461468.

Scanlon, M. J. and Norton, R. S. (1994) Multiple conformation of the sea anemone polypeptide anthopleurin-A in solution. Protein Sci. 3, 1121-1124.

Schbnbrunner, E. R. and Schmid, F. X. (1992) Peptidyl-prolyl cis-trans isomerase improves the efficiency of protein disulfide isomerase as a catalyst of protein folding. Proc. natn. Acad. Sci. U.S.A. 89, 451S-4513.

Smith, L. A. and Schmidt, J. J. (1990) Cloning and nucleotide sequences of crotamine genes. Toxicon 28,575585. States, D. J., Haberkom, R. A. and Ruben, D. J. (1982) A two-dimensional nuclear Overhauser experiment with

pure absorption phase in four quandrants. J. Magn. Reson. 48, 286292. Teno, A. M., Vieira, C. A., Santoro, M. M., Neves, A. G. A. and Giglio, J. R. (1990) Interchain disulfide bonds

in crotamine self-association. J. Biochem. 107, 821-825. Utaisincharoen, P., Baker, B. and Tu, A. T. (1991) Binding of myotoxin a to sarcoplasmic reticulum

CaZ+-ATPase: a structural study. Biochemistry 30, 8211-8216. Voloe. P.. Damiani. E.. Maurer. A. and Tu. A. T. (1986) Interaction of mvotoxin a with the Ca2+-ATPase of

skeletal muscle sarcoplasmic reticulum. Arch. B&hem. Biophys. 246, 90-97. Werner, J. M., Breeze, A. L., Kara, B., Rosenbrock, G., Boyd, J., Soffe, N. and Campbell, I. D. (1994) Secondary

structure and backbone dynamics of human granulocyte colony-stimulating factor in solution. Biochemistry 33, 7184-7192.

Wiithrich, K. (1986) Nh4R of Proteins and Nucleic Acids, Parts I, II, and IV. New York: Wiley-Interscience. Wutte, A., Giibitz, G., Friebe, S. and Krauss, G.-J. (1994) High-performance liquid chromatography of cis-trans

isomers of proline-containing dipeptides: III. Comparative studies with different stationary phases. J. Chromar. A. 677, 186-191.

Yudkowsky, M. L., Beech, J. and Fletcher, J. E. (1994) Myotoxin-a reduces the threshold for calcium-induced calcium release in skeletal muscle. Toxicon 32, 273-278.

Date post:	12-Nov-2023
Category:	Documents
Upload:	independent
View:	0 times
Download:	0 times

Evidence for isomerization in myotoxin a from the prairie rattlesnake (Crotalus viridis viridis)

Documents