Spectroscopic determinations of enzyme-catalyzed reactions at subzero temperatures

AX.\,,-.-‘1’,~‘.\1,

I~IOC’HEhlISTRY

57. 555-563

(1974)

Spectroscopic Determinations Reactions at Subzero

of Enzyme-Catalyzed Temperatures

The direct spectroscopic determination of enzyme-substrate intermediates during the course of some enzyme-catalyzed reactions can he performt~tl in niiscd solvents at subzero temperatures (1) but necessitates sl‘ecial c,qnipment of rommercial sljectrophotometers. This equipment must, sat,isfy the following requirements: (1 1 l’roduction of cryogenic tcml)c’ratures down to -8O-100°C; (21 tight control of the temperature of the‘ experimental samples; and 131 prevention of water condensation and iccx formation on cell walls, wiiitlowS , and optical 1~~~s involved in any spectrophotometer. Since many enzyme-substrate intermediates could b(x cletected by their characteristic nbs.orption sl)ertra, we equipped two commercial spectrophotometers of orclinary speed of response in this Iahoratory and will describe the al)pnratus then adapted to a Beckman Acta III and an Aminco-C’hnnre T)W2. Overall llerformances ITill be givcln and discussed, as well as home t,\-l)icnl spectra of ligandized forms of hemoproteins and kinetic recortlings of an enzyme-catalyzed reaction obtainc~cl in these conditions at. subzc~o teml~erntures.

The c’cluipment we used consists of three main parth: a crpogenic-temperaturcx production unit, a teml’erature-regulation device, and an adal)terl cell holder and sample compartment. The cell (glass or quartz, 1 cm optical path length) containing the saml)lc is plnced into a metallic ccl11 hol(ler) thermostated by a tc,ml’craturc-corltrollc~l gaseolls-nitrogen circulation. In order to avoid con(lcn~ation and ice formation that appear

51iO

MAUREL,

TRAVERS

ASD

DOUZOU

the cell holder is isolated into a on the cell walls at low temperaturcr, sample compartment overpressurized with dry nitrogen. CryoyeGc-Temperature Prodwtion Unit and Regulation Device. A general description is given in Fig. 1. a dry gaseous-nitrogen circulation (from gas container G), whose delivery can be varied between 0 and 50 liter/min by means of a flow meter (FM,) (Airliquide Dynamal) , is bubbled into liquid nitrogen, contained in a dewar (Dj (LDlO Union Carbide). It is then transferred under vacuum [by vacuum-tube transfer (VTT) 1 toward the cell holders (SCH, RCH), and heated as required by an electrical resistance (R 1, variable between 0 and 200 W, connected through an alternostat (AT) to a temperature regulator (R.G,. The tempcrature regulator (Barber Colmann Solid State, 540 series) is designed to accept the output’ of a standard Chromel-Alumcl thermocouple (TC) (Philips Industrie, S.A., type 2 ,4BI 025)) measuring the tcmperaturc of the circulating nitrogen after passing t,hrough the sample ccl1 holder. Then to obtain temperature rcgulntion, one needs only to select a desired temperature on thr temperature regulator set point dial, suitahlc nitrogen circulation delivery, and heating resistance. The temperature of the sample and reference is ciircctly measured into the cells with Chromclillume1 thermocouples (STC, RTC) conncctcd to a galvanometer (GL) (Sefram-Verivat) Cell Holders and Salr~ple C’ompartment. The structure of the various commercial spectrophotomcters being very different, it’ is necessary to adapt the cell holder to each particular type of apparatus. Beckman Acta III spectrophotometer. In this apparatus, t,he salnp]e and reference beams are separated by 12.7 cm. Consequently, two

SPECTROX’OPT

a. top

b-stde

vue

AT

SUBZERO

‘OB I I

,A /’

c-Front

vue

FIG. 2. Beckman

cell

357

TEMPERbTURES

holtlcr.

(‘onstruction

vue

B

,lcn

cl(~tails.

identical cell holders are used. Construction details are given in Fig. 2. Each cell holder consis& of an outer brass cylinder (A), inside which thermal-regulating nitrogen circulation is allowed. A second cylinder of the same material (B), whose inner section is square in order to adequately receive the cell, fits into the former. The B cylinder is movable around its axis, allowing selection of the correct cell position with Icgard to the optical beam. The two outer cylinders (A) are fixed on the floor of the compartment. They are connected together by a metallic T tube (TT), allowing an identical flow of nitrogen through each. Evacuation of the thermostating fluid is made by two independent exit, tubes on which two valves (V,. VZ) enable control of the circulation tltxlivery inside chili cell holder. It is thus possible to restore, if nccessarg, the thermal equilibrium between both sample and reference ~11:: by manipulating the valves. Two elliptical holes (11) are provided on each cylinder, in order to allow the optical beams (O.Bj to pass through the cells. In Fig. 2b and c, we show how the cell is placed into the cell holder in order to maximize hclnt exchanges, and how it can be extracted. Two metallic strips (Sp,) and (Sp2) are used for these purposts. Their curvature, acting as a laminated spring, maintains the cells against malls (I$‘, and W,) As with cylinders A and B, Sp, is provided with an elliptical hole permitting the I,assage of the optical beam. spl is used to extract the cell from the cell holder, as shown in Fig. 2~. In order to avoid damage of the cell walls, the cell-cell holder contact is effective only 011 the edges of the cell, cell-holder walls TT7, and Iv2 each

558

MACREL,

TRAVERS AND DOUZOU

- - --

--

FIG. 3. 13eckmnn adapted sanlple conlpartnlent

being provided with a channel, as shown in Fig. 2. This assembly is placed into an airt,ight compartment (STC) (Fig. 3) overpressurized with dry nitrogen (whose delivery is controlled by flow meter FM,) in order to avoid water condensation and ice formation on the cell walls which appears, directly or when opening the cell compartment for addition of reagent, at low temperature. This compartment is isolated from both opt,ical and electronic 1)art.s of the spectro])hotornct,er by means of four quartz windows (QWl;r Access to the cells is permitted via two circular holes, provided on the top wall of the compartment. As shown in Fig. 3, a vibrating stirrer 0%) (Ruchler Instrument) is mounted on the morable external top wall of the sample compartment. When used either for obtaining a good thermal homogeneity nithin the sample or for mixing after addition of a reagent, it is pushed down in order to work on the whole volume of the liquid. At rest, a spiral spring (SSP) brings it to an upper position, avoiding any interaction between the stirrer extremity and the optical beam. du~i~~co-Chance DlY2 spectrophotometer. In contrast to the I)revious apparatus, the sample and reference beams are 1.5 cm away from each

SPECTROSCOPI-

AT

SIBZERO

TEMI’ERATCRES

!

I

.SP, .SP,

-top

vue

SC,

_ j

-.

-front

vue 1 cm--

FIG.

-2. Aminc,o

cell

holder.

C’onstruction

details.

other. This makes it possible to plncc sample :md referenre cells into the wme cell holder. h detnilctl description is given in Fig. 4. The cell holder is made with :t cubic block of brass l~roriclcd with two cell chambers. inside which nitrogen circulation is nllo~yed. Proximity of the (~(~11~anal thermal incrtin of the ccl1 holtlcr enable thermal equilibrium Ixkww~ both cells to be easily obt.ained. Four elliptical holes (h) permit, tlrc optical beam (0.13) to pa:‘-ib through the sample and reference cells (SC’ :x111 RC‘ I. The wmaining parts of the cquilw~ent, (cell compartment, stirwr) , 3s well ns tllcd method for placing the cell into the cell holder, are the same as those described previously. PF:IIFOI~2IS~~‘l~‘-: /

1

.I

Twlperntzcre ConSrol. The acccsGble range of temperature that such nu cquipinent~ nllows stretches from -120 to 100°C. Calibration of the

560

MAUREL,

TRAVERS

AND

DOUZOU

assembly thermocouple-galvanometer leads to an absolute uncertainty of +-O.l”C on the measured temperature. This precision is sufficient for most of our experiments. Moreover, we have verified the stability and homogeneity of temperature in both cells by replacing the galvanometer by a Beckman Recorder. When temperature regulation is obtained, no temperature variation can be detected, either with time or within the total volume of the sample, precision being better than -tO.l”C. In Fig. 5, we present a few recordings obtained with the Beckman equipment when raising or decreasing the temperature. Three typical cases have been chosen: both large and small decreases (-75 and -20°C) and a small increase ( + 15°C). It appears that temperature equilibrium inside either sample or reference cells is obtained within a reasonably short time. When using the Aminco-Chance equipment<, where only one cell holder has to be thermostated, these times of equilibrium are slightly shorter. Differential Spectra. As an application of differential spectra at low temperature, the displacement by temperature variations of the equilibrium between two ligandized forms of reduced cytochrome P-450 has been investigated. The addition of ethylisocyanide (EtCN) to a suspension of reduced rat liver microsomes gives rise to two ligandized forms of cytochrome

0

4

8

12

hme (mn)

FIG. 5. Recordings of temperature vzuiation with time inside the Beckman N? delivery : (1) 30 liters min-‘, R = 50% of its total value; (2) 20 liters R = 50% of its total value; (3) 10 liters min-I, R = 65% of its total value.

cell. min-‘,

SPECTROSCOPY

AT

SUBZERO

TEhlPERATURES

561

P-450 which are in equilibrium. The differential spectra between cyt*Fe’+EtCN and cyt.Fe’+ presents two absorption maxims at 430 and 455 nm. It is well-known that this equilibrium can be displaced by either pH or ionic strength variations (,2,3). The possibility of recording such differential spectra as a function of t,empernture, from 20 down to -50°C in water-glycerol mixture (30-70/ c~ m volume), leads us to show that such an equilibrium is displaced by temperature variation. These recordings are shown in Fig. 6, where we can see that, the “455-nm form” is reversibly converted to the “430-nm form:” the isobest.ic point lying at 436 nm. A d&ailed investigation of this process is now being carried out in this laboratory. Steady-Stnte lii?lelics. We measured the steady-state reaction rate of the horseradish peroxidase (HRP) catalyzed oxidation of guaiacol by hydrogen peroxide as a function of temperature from 20 down to -65’C in a hydroorganic solvent (water-methanol, 40-607~ in volume). Horseradish perosidase was purchased from Sigma Chemical Co. (type VI) and used without any other purification; substrates and methanol are from Merck. Thc~ reaction rate is det8ermined with the Aminco-Chance Spectrophotometer DWZ by the increase with time of the absorption at 430 nm due to guaiacol oxidation to form tetrnguainocoquinone. Enzyme con-

T 01

1

,I

FII;. 6. Differentinl ~pwt~~n lwtwcen cyt .Fc”-ELCN and cyt.Fe’+ nt, tlifferrnt Icmperature~. Rat liver micro;;omr cyt.P-450 is suspended in a 0.02 M phosphate l~uffrr, pH 8. glycerol misturr (30-70 c/L in volume), reduced by exces of dithionite, and placed in both sample and rrferencc ~11s. EtJhylisoryanidc is then addrd in the samplr cell, ib final concentrntion being 5.10.‘M. (1) 21”C, (2) 8°C. (3) -7”C, (4) -2O”C, (5) -3O”C, (6) -39”C, (7) -50°C.

MAUREL,

FIG.

peratures.

7. steady-state Horseradish

TRAVERS

kinetic curves of perosidase = 2.10m”

AND

DOUZOU

a perosidnse phosphate

M,

reaction al) different buffer 5.1Om” M, pH 7.

tem-

c&ration varies from lo-‘; to 1P M, depending on the temperature of expcrimcntatior1, in order to get a convenient reaction rat’e for the particular measuring method used. Roth substrates are used in their saturation range-guaiacol 2 X lo-” M hydrogc>n peroxide 2.5 X lo-” M. The overall precision on the reaction velocity is about 5% in the whole temperature range. Direct curves of increase of absorption with time are shown in Fig. 7 for various temperatures between -30 and -65°C. The initial transient state (part OAj is smoothly followed by a linear part (AB) responsible for the establishment of a steady st’ate. It appears that, at low temperatures, steady state occurs rather a long time after beginning the experiment. These results enable UP to determine an sctivation energy of 12.0 + 1 kcal/mole for that reaction by using the Arrhenius equation. It must) bc emphasized that the Arrhenius plot we obtained is linear over the whole range of experimental temperatures. Systematic studies of the Ion-tetnpcrature effect on kinetics and, subsequently, mechanisms of enzyme reactions, are now under estensive investigation in this laboratory. PROBLEMS

Substrate Addition CL& Stiwing. In some cases (particularly in kinet,ic studies), introduction of one or several reagents into the cell and consequent stirring of the mixture are necessary, and lcnd to both thermal

SPECTROSCOPS

AT

SUBZERO

TEMPERATL-RES

563

nonhomogeneity and a small momentary rise of temperature inside the sample. We have rcducctl these effects by adding a very small quantit8y of reactants (a few mirrolitrts by means of a mic*rofGpett,e I, and by using a low thermal inertia stirrer pernwncntly maintained ill tllc cell at the s:tmple tenilwraturc. IIowwc~r, our csperimc~nts show that, at wfficientJy low temperatures \wlwre these cficctr are ni:tsimal 1, the rate of reactions are very low, ant1 in most c:k+s thermal cquililkum is wstored far heforc steady state aIq)e:trs. Cklierwiw, a stopp~l-flow apparatus adnlkccl to the s:ame range of subzero tcmlwnturc~ ant1 rwcntlp dcwribed (4) could 1~ used to perform kinetic studies.

1 Douzou. 2.

IMM,

I'.,

3.

h41.

E'..

4.

HIJI

130~

P. (1973) ilId. Cell. f~iochcm. 1, 1. 4x1) SATQ, R. (1966) /&rhcw. ~?iophys. 12~s. Commlrtl. 23, 5. .4ND &TO, 8. (l(368). sf. &ochenl. (Tok{/o) 63, 270. Ho.\. G.. AND Douzou. P. (1973) Annl. Riochem. 51, 127.