O'Donnell and McGeeney: lodination of cramylase using solid state lactoperoxidase 551

Z. Klin. Chem. Klin. Biochem.12. Jg. 1974, S. 551-557

lodination of -Amylase using Solid State Lactoperoxidase1)

By Mary D. O'Donnell2) and K. F. McGeeney

Department of Medicine & Therapeutics, University College, Dublin 4

(Eingegangen am 19. Juni/31. Juli 1974)

A radioiodination method using bovine lactoperoxidase covalently coupled with Sepharose-4B, was successfully applied to a-amylase.Unlike some chemical iodination procedures, the lactoperoxidase method appeared to be quite gentle, employing only 5—10Na125I and traces of hydrogen peroxide as oxidising agent. The catalytic activity of the labelled enzyme appeared to depend on thetotal iodide to amylase molar ratio. Ratios greater than 0.6 reduced the catalytic activity. There was no loss of enzyme activity wheniodinated at ratios up to 0.6. Amylase labelled under these conditions appeared suitable for metabolic studies. When canine plasmaamylase concentration was artificially raised (5—10-fold), the disappearance curves of radioactivity and amylase activity were parallel.Tracer studies in dogs are also described.

Unter Verwendung einer an Sepharose 4B kovalent gebundenen Rinder-Lactoperoxidase ist es gelungen, Amylase radioaktiv zu jodie-ren. Im Vergleich zum chemischen Verfahren verläuft die Lactoperoxidase Methode unter schonenden Bedingungen. Lediglich5-10 Na125J und Spuren von Wasserstoffperoxid als Oxidationsmittel werden benötigt. Die katalytische Aktivität des markiertenEnzyms scheint von dem molaren Verhältnis des gesamten Jodids zur Amylase abhängig zu sein. Ein Verhältnis größer als 0,6 vermin-dert die kataiytische Aktivität. Bei einem molaren Verhältnis unter 0,6 trat kein Aktivitätsverlust ein. Auf diese Weise markierte Amy-lase scheint für Stoffwechseluntersuchungen geeignet zu sein. Nach experimenteller Erhöhung der Konzentration der Plasma-Amylase·bei Hunden auf den 5-10fachen Normalwert verliefen die Abnahmekurven von Radioaktivität und Amylase-Aktivität parallel. Tracer-Untersuchungen an Hunden werden beschrieben.

There are few reports in the literature on the successfullabelling of enzymes without loss of catalytic activity.In the case of a-amylase (EC 3.2.1.1) the iodine mono-chloride method (1) caused severe loss of enzyme acti-vity (2, 3). The method of Greenwood et al. (4) has beenused for many enzymes (5—9). Chloramine-T can causeoxidation of amino acids other than tyrosine resultingin protein denaturation (10) and loss of catalytic activity(6). These disadvantages gave impetus to the search fora more gentle means of radioiodination. The electro-lytic method (11) is reported to be very mild. It wasused for a-amylase (3,12) with minimal loss of enzymeactivity.

Recently there have been several reports of successfuliodination of proteins by means of the enzyme lacto-peroxidase (EC 1.11.1.7) (10, 13,14), It was used bythese workers in soluble form in trace labelling albuminand immunogjobulins. Soluble lactoperoxidase has alsobeen used in labelling proteins on the outer surfaces ofmammalian cells without loss of cell viability (13,15,16). The method can also give high specific activitiessuitable for radioimmunoassay (17,18).An improved method employing solid state lactoperoxi-dase has been described (19,20). By covalently couplingthe enzyme to an insoluble matrix such as Sepharose-4B

it can be removed from the iodination reaction mixturesimply by centrifugation. Moreover, very low concen-trations of oxidising agent i.e. hydrogen peroxide andradioactivity are required. For these reasons, the methodwas studied for the iodination of -amylase as describedbelow.

Materials

EnzymesBovine lactoperoxidase and porcine -amylase were purchasedfrom Sigma Chemical Co., London, U.K. Canine amylase waspurified from canine pancreatic juice (1000 U/mg protein).

ChemicalsAll reagents were of Analar grade except the following whichwere obtained from the sources indicated: bovine albumin(fraction V), sodium azide and glycine, Sigma Chemical Co.,London, U. K.; PHADEBAS blue starch tablets, blue dextranand cyanogen bromide activated Sepharose 4 B, Pharmacia,London, U.K.; Bio-Gel P100, (50-150 mesh), -Rad Labora-tories, Richmond, California; hydrogen peroxide and sodiummetabisulphite, Hopkin & Williams, Chad well Heath, U.K.;Na12SI, Amersham, U.K.; human ^-globulin, Mann Research

*) Presented at the 1st European Congress of Clinical Chemistryin Munich (22.-26. 5.1974).

2) Supported by the Medical Research Council of Ireland.

Z. Klin. Chem. Klin. Biochem. / 12. Jahrg. 1974 / Heft 12

552 O'Donnell and McGeeney: lodination of o-amylase using solid state lactoperoxidase

Laboratories, New York; Ζ,-cysteine hydrochloride, BritishDrug Houses, Poole, U.K.; 131I-labeUed albumin, Ames Com-pany, Miles Laboratories, Slough, Buckinghamshire, U.K.;chloramine-T, May & Baker, Dagenham, U.K.

ProceduresEffect of chloramine-T on a-amylase action75 Mg porcine o-amylase was incubated with increasing quantities(2-80 Mg) of chloramine-T in 50 μΐ 0.05 mol/1 phosphatebuffer containing 50 mmol/1 Nad and 0.5 mmol/1 CaCl2. Theincubation was for 1 minute at 22° C. 0.5 ml of sodium meta-bisulphite (1 mg/ml) was then added, the mixture dialysed andthe catalytic activity of the enzyme determined after suitabledilution. Comparison was made with a control without chlor-amine-T pre-treatment, the value of which was taken as 100 %activity. The effect of time on the reaction was studied asfollows. 75 μg amylase was incubated with 80 μg chloramine-Tas above. Aliquots of the reaction were removed at 10, 25, 35, 45and 60 seconds and added to 0.5 ml of sodium metabisulphiteprior to dialysis and analysis.

Preparation of solid-state lactoperoxidaseThis procedure was performed as described by David (19). Byrecording the extinction at 412 nm before and after coupling,the enzyme was calculated to be coupled to the gel to a finalconcentration of 2.8 mg/ml of settled beads. The Sepharosebound lactoperoxidase was stored at pH 7.4 in 0.01 mol/1 phos-phate buffered saline in a volume such that 1.0 ml of suspensioncontained 1 mg of lactoperoxidase.

lodinations:The procedure of David (19) was followed. All iodinations wereperformed using the following concentrations of reactants unlessotherwise indicated: lactoperoxidase, 10 Mg/ml; potassiumiodide, 10 μηιοΐ/ΐ; hydrogen peroxide, 90 μιηοΐ/ΐ; phosphatebuffer, 0.05 mol/1 containing 0.05 mol/1 NaCl and 0.5 mmol/1CaCl2, pH 7.0; Na125I containing about 107 c.p.m. (i.e. ~ 5 μΟί).Following iodination, and the removal of the Sepharose boundlactoperoxidase by centrifugation (3000 g, S min), the super-natant was dialysed in Visking tubing against several changesof phosphate buffer pH 7.0 until the counts in the externalfluid disappeared. In some cases, i.e. when iodination reactionvolumes of 0.1 ml or 0.2 ml were used and in the time curve ex-periments the reaction was stopped with 0.8 ml of 5 mmol/1cysteine (10) before centrifugation and dialysis.The course of the iodination reaction was followed by precipi-tating aliquots of the reaction mixture, with trichloroaceticacid (19) at timed intervals up to 30 minutes. The radioactivityin the supernatant and precipitate was then counted in a Tra-cerlab gamma/guard 150 spectro/matic counter (ICN, U.K.).The percentage precipitability of the radioiodine in the super-natant after removal of the Sepharose bound lactoperoxidasewas determined. Per cent loss of the 125I counts due to "self-iodination" of the lactoperoxidase (at t = 30 min) was calcu-lated from the total iodine remaining in the supernatant andprecipitate as compared to the zero time sample. Per cent in-corporation of counts into α-amylase was based on the totalradioactivity added at zero time. 125I utilization (at t30) = %"self-iodination" + % 12SI incorporation (20). The free 12SI (%)of Table 1 (at t30) = non utilized 125I, i.e. 100-125I utilized.

Gel filtrationA Bio-Gel P-100 column (2 X 62 cm) was equilibrated andeluted with 100 mmol/1 NaCl containing 0.5 mmol/1 CaCl?and 10 μπιοΐ/ΐ albumin. Aliquots of iodinated enzyme dilutedto 1.0 ml with elution fluid were applied to the column, andfractions (2.25 ml) were collected at a flow rate 12-15 ml/hour.

Comparison of labelled and unlabelled amylase in substratebinding:25 mU of labelled amylase was added to increasing amounts ofblue starch substrate in 1.0 ml of phosphate buffered saline *pH 7.0 at room temperature. After 2 minutes each tube wascentrifuged at 2000 g (5 minutes) and the substrate washed

and centrifuged 5 times with 1.0 ml aliquots of the samebuffer. The washed blue starch substrate and pooled super-natants were then counted and the bound enzyme (%) calcu-lated. The substrate binding of unlabelled amylase (52 mU)was studied by measuring enzyme activity with increasingsubstrate concentration.

Experimental AnimalsThree conscious female dogs (Canis familiaris) weighing 12-15 kgwere used. Urine samples were obtained vifycatheter insertedinto the bladder prior1 to the experiment. Blood sampling was .facilitated by venous catheter in the leg or neck, fitted one dayprior to the experiment. The animals were fed potassium iodidefor two days beforehand in order to block the thyroid (21).

Comparison of the physiologic behaviour of radiolabelled and un-labelled amylaseThe serum disappearance curves and renal clearances of 125l·labelled and unlabelled canine amylase were determined inthree dogs. Labelled amylase and sufficient cold amylase toraise the serum amylase level 5—10-fold was injected intrave-nously in 10 ml of physiological saline over a 1-2 minuteperiod. Six heparinised blood samples were taken over the firsthour and then at hourly intervals up to 6 hours. Each bloodsample was replaced with a similar volume of physiologicalsaline to maintain the blood volume constant. Urine was collec-ted every 60 minutes over a 6 hour period. Each serum andurine sample was analysed for radioactivity, amylase activityand creatinine. Serum disappearance curves were constructedand renal clearance of amylase and radioactivity calculated (12)and expressed relative to the creatinine clearance.

Tracer studiesSerum disappearance curves and renal clearances were studiedat normal serum amylase levels after injection of tracer quanti-ties (15 U) i.e. ~ 1 % of total serum amylase. Samples werecollected as described above.

Assay methodsα-Amylase was measured by the method of Ceska et al. (22)and incorporating 7 μηιοΙ/1 albumin in the reaction mixture(23). Urine creatinine was measured by the method ofBonsnes& Taussky (24) and plasma creatinine by a modification of thesame method (25). Haematocrits of all blood samples weremeasured. Blood volumes were measured by intravenous injec-tion of 131I-labelled albumin (~ 1 μΟϊ) and measuring the degreeof dilution using a Volemetron (Atorhiiim, Billerica, Mass.)apparatus. This injection was administered with the α-amyl se.The interference of these 131I counts with trie 125Habelledamylase counts was minimal and was calculated via a standardcurve. An appropriate correction factor was then applied. Plasmavolume was calculated from the blood volume and haematocritvalues. The rate constants were calculated according to Matthews(26).

Results

Effect of chloramine-TOn a-amylase activity<*Amylase was found to be very sensitive to chlor-amine-T. The results of a 1 minute incubation of α- my-lase with varying concentrations of chloramine-T isshown in Figure l . The addition of 50 μg chloramine^T to 75 μ§ amylase/50 μΐ, reduced the enzyme activityto 39.3 % of the control, while 80 Mg almost abolishedthe catalytic activity. A time curve relating the effectof chloramine-T (80 #g) ori α-amyl se with time isshown in Figure Ib. Within 10 seconds 60 % of theenzyme activity was lost. This loss was progressive;over 90 % of the catalytic activity was destroyed within1 minute.

Z. Klin. Chem. Klin. Biochem. / 12. Jahrg. 1974 / Heft 12

O'Donnell and McGeeney: lodination of o-amy l se using solid state lactoperoxidase 553

100

_ 80. Ei

.f 601g 40M*

^ 20

o

o~o^ 100«\

\ 80\\ 60

\V / n40\\\ 20\

| | 1 J

>- David (19). One would have expected that at highι amylase concentrations less 125I would be lost. Instead{· we see that at 0. 1 5 mg amylase/ml, 2 1 .4 % 125I is lost'\ while at 1.3 mg/ml 45 % is 'lost'. The reason for this is"\ not clear at present. However, the total 125I lost i.e. due

^ to self-iodination by sepharose lactoperoxidase plus\^ free 125I appears to be constant. This amounts to 52 %

\^ at 0.15 mg/ml and 55 % at 1.3 mg/ml (Table 1).b x^ It is apparent from Table 2 that when the amylase con-

I « I 1 . . . . « . · . « / . « . .1 rf; rjj g« on Q 20 40 60 cenuauon is Kepi mgn ^i.e. oy reducing me reactionChloramine-T [uq] Time [s] volumes), I incorporation decreases with decreasing

Fig. 1. (a) Effect of Chloramine-T (l min, 22° C) on o-amylaseactivity (75 Mg enzyme protein/50 μΐ).

(b) Effect of Chloramine-T (80 Mg) on α-amylase activityas a function of time (75 Mg enzyme protein/50 μΐ).

lodination ofot-amylase by solid state lactoperoxidaseThe concentrations of lactoperoxidase, KI, H2O2 andbuffer system used were those of David (19). The effi-ciency of iodination of α-amylase at a concentration of1.3 mg/ml (Table 1) and the rate of iodine incorporation(Fig. 2) were similar to those obtained with IgG (19)at a comparable protein concentration. The effect ofvarying amylase concentration is also shown in Table 1.The fact that 12SI loss' due to self-iodination of Sepha-roselactoperoxidase is lower at low amylase concentra-tions is surprising and differs from the findings of

mass of amylase. There is 45 % incorporation at 1.3 mg/ml with only 33.3 % at 0.15 mg/0.1 ml. This effect isnot apparent at low amylase concentrations (Table 1).A corresponding reduction in % precipitability of io-dinated amylase was found, 42.4 % when iodinated at0.15 mg/0.1 ml (Table 2) compared with 61.5 % at0.15 mg/ml (Table 1). The higher degree of 12SI incor-poration at low amylase concentrations could be dueto a structural change in the amylase molecule depen-ding on the concentration. At low concentrations thisconformational change may make more tyrosine residuesaccessible for iodination.

Effect of iodination on catalytic activity ofa-amylaseWorking with an amylase concentration of 1.3 mg/mlthere appeared to be no loss in enzyme activity (Table 3).

Tab. 1. lodination of α-amylase by lactoperoxidase at differentamylase concentrations 10 Mg/ml lactoperoxidase;10 Mmol/1 KI; 5 MCi125!; 90 Mmol/1 H2O2; phosphatebuffered saline, pH 7.O.

Tab. 2. lodination of α-amylase by sepharose-bound lactoperoxi-dase at constant amylase concentrations. 10 Mg/ml lacto-peroxidase; 10 Mmol/1 KI; 5 MCi125!; 90 Mmol/1; phos-phate buffered saline pH 7.O.

Amylase

[mg/ml]

0.150.31.3

C13CCOOHprecipita-b ity[%]

61.56681.6

125I -incor-poration

[%]

4847.54?

125I 'loss'

[%}

21.428.445

125Ifree

[%}

30.624.110

Total 12SIutilization

[%}

69.475.990

Aiuyiaac;

0.15 mg/O.lml0.3 mg/0.2ml1.3 mg/ml

v,i3VxVxvyvy«.precipita-bility

42.4

47.7

81.6

poration

33.3

42

45

\yO\

21.1

17

45

utilization

54.4

59

90

100

• 80

I 60I

•

' 40L

: 20

10 15 20Time [min]

I 125 30

Fig. 2. Time curve showing 125I incorporation into porcineo-amylase (0.3 mg/0.2 ml) by Sepharose-bound lacto-peroxidase.

Tab. 3. Dependence of amylase activity on iodide/amylasemolar ratio during iodination by lactoperoxidase.10 Mg/ml lactoperoxidase; 10 Mmol/1 KI; 90 Mmol/1H2O2; 5 MCi12*!; phosphate buffered saline, pH 7.O.

Amylase

[mg]

0.150.30.651.30.150.075

Volume

[ml]

1.01.01.01.00.10.1

Iodide/ Amy-lase ratio

3.31.660.80.50.330.6

EnzymeActivity

[%]

32.55575

100100100

Radioacti-vity[counts/min · U]

12,50050,100

571,000*

Z. Klin. Chem. Klin. Biochem. / 12. Jahrg. 1974 / Heft 12 42

554 O'Donnell and McGeeney: lodination of α-amylase using solid state lactoperoxidase

However, iodination at a concentration of 0.15 mg/mlcaused a marked reduction in enzyme activity to 32.5 %.Even at 0.65 mg/ml enzyme activity was reduced to75 %.The reason for this reduction in catalytic activity wasthen investigated. Initial experiments showed that theenzyme at a concentration of 0.15 mg/ml was not sen-sitive to 90 jumol/l H2O2 on its own. Neither was itsensitive to the other components of the reaction, i.e.KI, 125I, or Sepharose bound lactoperoxidase since acontrol experiment, without the H2O2, had no effecton enzyme activity. The effect of temperature on theiodination was investigated since it was thought thatiodination at 0°C might prevent denaturation at lowamylase concentrations. It is shown in Table 4 thatiodination at 0°C had a somewhat more adverse effect.The loss in catalytic activity was greater than wheniodinated at room temperature. However, low tempe-rature had no great effect on iodination efficiency as isevident from the figures for 125I incorporation (Table 4).

The loss of enzyme activity appears to be a function ofthe iodide/amylase molar ratio (Table 3). In the casewhere 32.5 % enzyme activity remains a ratio of 3.3 ispresent. At an iodide/amylase value of 0.8, enzyme acti-vity is 75 %, while at a ratio of 0.5 there is 100 % cata-lytic activity. To iodinate 0.15 mg of amylase, therefore,without losing activity the enzyme concentration mustbe kept high relative to the iodide concentration. Thisis done by reducing the reaction volume containing0.15 mg amylase to 0.1 ml. The iodide/amylase ratio isnow reduced to 0.33 giving full enzymatic activity.Ratios > 0.8 have a deteriorating effect. By varying thequantity of 125I, high specific activities can be obtained,e.g. using 50 μ€ί 12SI and 0.075 mg amylase/0.1 ml aspecific activity of 571,000 cpm/unit enzyme was ob-tained (Table 3). Such apecific activities are desirablefor tracer studies. α-Amylase, purified from caninepancreatic juice, behaved similarly to the porcine enzymewith regard to 125I-incorporation, precipitability andsensitivity to high iodide/amylase ratios.

Gel filtration of labelled amylaseThe behaviour of labelled amylase (iodinated at iodide/amylase ratio of 0.33) on a Bio-Gel P-100 gel filtration

Tab. 4. Effect of temperature on iodination reactionslactoperoxidase; 10 μιηοΐ/l ΚΙ; 90 μηιοΐ/l H2O2; 5 MCi125I;phosphate buffered saline, pH 7.O.

column is shown in Figure 3. Its elution profile coin-cided with enzyme activity. When iodinations wereperformed at high iodide/ amylase ratios causing cata-lytic activity to be reduced, the radioactive peak on gelfiltration emerged slightly to the left of the main en-zyme peak. This indicates that loss in activity is accom-panied by a change in structure of the labelled mole-cules, which is detectable on gel filtration.

Comparison of labelled and unlabelled amylase insubstrate bindingThe ability of labelled amylase to combine with bluestarch was studied as described in Procedures. Figure 4,curve (a) shows a plot of the % labelled enzyme boundto substrate vs. substrate concentration. A parallel curve(b) was obtained for unlabelled enzyme. The parallelcurves indicate that the labelled product is combiningwith its substrate in a similar manner to the unlabelledenzyme.

Amylase

[mg/ml]

0.15

0.3

Temperature

[°C]

220220

C13CCOOHprecipit-ability[%]

61.5686650.3

12SI incor-poration

[%]

484647.541.3

EnzymeActivity

1%)

32.520 -5550

2800r

-=2400

^ 2-000J!eSi 1600

.1200

' 800

400

70

60S.i

50 |H

405=>ε30

10

32 40 48 56Fraction number

64 72

Fig. 3. Elution profile of 125I-labelled amylase (o) and enzymeactivity (·) on Bio-Gel PI00 (Vt157 ml; V046 ml; frac-tion vol 2.25 ml; Eluent 0.1 mol/1 Nad, 0.5 mmol/1CaCl2 and 10 μιηοΐ/ΐ albumin). *

20 40 60Blue starch [mg/tubel

Fig. 4. Sirbstrate^bound i25I-labelied amylase vs. substrate con-centration (a) compared with enzyme activity vs. sut*strate concentration (b).

Z. Klin. Chem. Klin. Bipchem. / 1*2. Jahrg. 1974 / Heft 12

O'Donnell and McGeeney: lodination of oramyl se using solid state lactoperoxidase 555

Comparison of plasma disappearance curves of labelledand unlabelled amylaseThe disappearance curves of radioactive amylase andamylase activity were compared in dogs after intravenousinjection of a mixture of labelled and unlabelled enzymeas described in Procedures. In each of three studies,radioactivity disappeared from the plasma at almost thesame rate as did amylase activity until normal serumamylase levels were approached. The curves could beresolved into at least two exponential components (26).The typical result is shown in Figure 5. This iodinatedamylase appeared to trace amylase catabolism sincethe specific activity of the serum (cpm/amylase activity)remained constant over the 6 hours. If an appreciabledifference existed in the rates of catabolism of thelabelled and unlabelled enzyme, the specific activitywould be expected to rise or fall above that injected.The fact that it remained constant is satisfactory sincemetabolic turnover studies require that the iodinated andnon-iodinated molecules are catabolised and excretedat the same rate (21, 26).

Tracer studiesTracer quantities of 125I-labelled amylase were admini-stered intravenously to two dogs. The typical result isshown in Figure 6 a. The % of dose remaining in theplasma is plotted against time and represented by lineQp. Urine samples were also measured for radioactivityand from each figure the amount of label remaining inthe body at that time could be calculated. The quantityof enzyme remaining was then plotted as a function of

1000,000

_ 500,000

"•S 100,000

I0:50,000

10,000

5,000

1,000 gj

50051

Ο 1 2 3 A 5 6Time [h]

Fig. 5. Simultaneously measured plasma disappearance curves foramylase activity and redioactivity in the dog after i.v.injection of labelled and unlabelled enzyme. The solidline represents measured values, while the dotted line iscomputed by extrapolating the steady-state fall-off backto zero time and subtracting the resulting line from theobserved data.

1008060

40

20

•-S

2̂ 100I 80ε 60

40

20

2 3 4Time [h]

0 1 2 3 4 5 6Time [h]

Fig. 6. (a) 125I-labelled amylase turnover data Qp = plasmacurve, Qr = total body curve i.e. total dose minusurine, Qe = extravascular curve calculated from Qr—Qp.

(b) Plasma curve (·) for amylase turnover as in (a). Thedotted line (o) is obtained by extrapolating the steady-state fall-off back to zero time and subtracting theresulting line from the observed data.

time. This line is represented as Qr in Figure 6 a. Thelinearity of this total body curve indicates that the label-'led enzyme is being removed at a constant rate at leastover 6 hours. Ideally the experiment should be con-tinued over several days and the amount of tracerexcreted in the faeces determined also.

Knowing the quantity of enzyme in the plasma and thetotal amount in the body, the difference must be extra-vascular. This curve is represented as Qe (Fig. 6 a). It canbe seen that after 6 hours ~ 20 % of the dose is still inthe plasma, and ~ 20 % has been excreted in the urine;this means that ~ 60 % has entered the extravascularregion. The plasma disappearance curves for both dogswere similar and again could be resolved into at leasttwo components. One such result, is shown in Figure 6b.Due to the rapid metabolic clearance of amylase (ti/2= 262 min; Table 5) identification of a third componentin the plasma disappearance curve would not be possiblewithout extending the experiment over a longer timeperiod.

The theory of tracer experiments using 13 ̂ -labelledplasma proteins was presented by Matthews (26). Ourresults appear to fit the simplest model, i.e. an opentwo-compartment system. The rate constants (Table 5)are similar to those obtained for the baboon (12). Theenzyme had a distribution half-life of 49.5 min and ametabolic half-life of 262 min (mean of two experi-

Z. Klin. Chem. Klin. Biochem. / 12. Jahrg. 1974 / Heft 12 42A

556 O'Donnell and McGeeney: lodination of o-amy l se it sing solid state iactoperoxidase

Tab. 5. Rate constants for plasma I25l-labelled amylase turnoverin the dog

distribution metabolicclearance

[min'1]-0.003185

[min"1]- 0.00987

[min'1!- 0.004095

[min]49.5

[min]262

ments). At raised plasma amylase levels the enzyme hada shorter distribution half-life, i.e. 23 min and a meta^bolic half-life of 234 min (mean of 3 experiments).

Renal ClearanceA summary of the data on amylase and creatinine ex-cretion at normal serum amylase levels is shown inTable 6. The ratio Cam/Ccr represents the rate at whichamylase was cleared relative to creatinine. When amylaseexcretion was measured as enzyme activity very lowvalues were obtained i.e. in the range 0.002—0.005 % forDog 1 and 0.11-0.15 % in the case of Dog 2. Whenamylase clearance was measured as radioactivity theCam/Ccr ratio reached a plateau of 5.3 % after 4 hoursin the case of Dog 1 and 4 % in the case of Dog 2. Aplot of the clearance of 125I counts and enzyme activityagainst time, both corrected for simultaneously meas-ured creatinine clearance, at normal serum amylaselevels (a) and at raised serum levels (b) in the same dog,is shown in Figure 7. Enzyme activity was minimalwhile 125I counts were excreted at a rate 3.5-5 % ofthe creatinine clearance. The 12SI counts excreted, inboth cases, were shown to be over 95 % dialysable,

Tab. 6. Data on amylase and creatinine excretion' in two tracerexperiments

Timeperiod

DOG10-1 h1-2 h2-3 h3-4 h4-5 h5-6 h

DOG20-1 h1-2 h2-3 h3-4 h4-5 h5-6 h

Serumamylase[U/1]

147514101385136013851410

193019001880186018201810

Urineamylase[U/l]

15.34.94.01.15

. 2.45.25

700520520740560270

m CT

0.0050.0020.0020.0030.0050.004

0.1320.1070.160.1530.1180.095

Γ* 125τ/*-am- V

1.332.223.55.255.35.1

0.871.833.43.45 ·3.94.6

2 3 4Time [h] ι

3Time -[h]

Fig. 7. Renal clearance of amylase activity (Cam) (o) andlabelled amylase, measured as radioactivity (o) in the dog,both corrected for simultaneously measured creatinineclearance (CCr) at normal plasma amylase levels (a) andat raised (5—10 fold) plasma amylase levels (b).

and were not precipitable with trichloroacetic acid; theurinary counts, therefore, must represent degraded amy-lase.

Discussion

The purpose of this investigation was to establish asuitable method for labelling α-amylase which fullypreserves the catalytic activity of the enzyme. Thechloramine-T method (4) has been used in labellingmany enzymes mostly for radioimmunoassay (5—9).As the immunological sites are usually different fromthe catalytic sites (5, 27, 28) it would be possible toachieve a labelled product suitable for radioimmuno-assay, with intact immunological sites, but with greatloss of conformity associated with alteration in catalyticactivity.

The loss of catalytic activity of α-amylase on treatmentwith chloramine-T (1 Mg/jug enzyme) has been demon-strated here. The method (4) would therefore be un-suitable in labelling α-amylase for in vivo studies dueto the associated conformity change. A similar sensi-tivity to chloramine-T was found in the case of fructose1,6 diphosphatase (6).

The main advantage of the Iactoperoxidase method isthat it can give a labelled amylase with 100 % catalyticactivity. The loss of enzyme activity obtained at highiodide/amylase molar ratios (> 0.8) was obviouslycaused by some product of the iodination reaction,probably molecular iodine; I2 has been shown to inhi-bit α-amylase by combining with internal sulphydrylgroups (2). Preservation of catalytic activity was pro-bably also facilitated by the low concentration of

Z. Klin. Chem. Klin. Biochern. / 12. Jahrg. 1974 / Heft 12

O'Donnell and McGeeney: lodination of α-amylase using solid state lactoperoxidase 557

hydrogen peroxide and radioactivity used. The fact thattyrosine residues are unlikely to be involved in a-amy-lase catalysis (29) is also fortunate. Since tyrosine mustform a complex with lactoperoxidase in the iodinationreaction (30) it is likely that tyrosine residues on theouter surface of the molecule only are labelled. Clearly,the large size of the lactoperoxidase would prevent itsbinding with internal tyrosine residues.

The variables studied in this investigation were amylaseconcentration and iodide/amylase molar ratios. Ideally,in order to determine optimal conditions for iodinationof a given protein, one should investigate the effectsof all parameters including concentrations of lacto-peroxidase, Η2θ2, ΚΙ, protein, iodide/protein ratio andbuffer system. Preliminary experiments established thatα-amylase was stable over a concentration range 0.15—1.5 mg/ml, in the presence of 10 μ§/πύ lactoperoxidase,90 μιηοΐ/ΐ H2O2,10 μιηοΐ/l ΚΙ, 5-10 μιηοΐ/l ΚΙ,5-10 μ€1125Ι in phosphate buffer pH 7.0. These werethe components for the iodination of IgG (19). Whilethe concentration of H2O2 used was optimal for lacto-peroxidase at pH 7.0 (30) it might be possible toincrease the efficiency of the iodination of α-amylaseby using a lower concentration of H2O2 and lactoperoxi-dase. The efficiency of iodination of IgG was increasedby using 18 μιηοΐ/ΐ Η2Ο2 and 2 μg/ml lactoperoxidase(20).

The lowest concentration of amylase at which iodinationwas attempted was 0.15 mg/ml. While some enzymes areunstable in very dilute solutions without the additionof carrier protein (31) the absence of carrier protein inthe iodination reaction did not, per se, cause inactiva-tion since the dilution of amylase was not high enoughto cause irreversible denaturation. As mentioned above,a control experiment showed that α-amylase, at a con-centration of 0.15 mg/ml for at least 30 minutes re-tained full catalytic activity. However, inactivation bydilution might be a factor in the case of other enzymes.

The use of insolubilised lactoperoxidase has a numberof advantages over the soluble enzyme. It can be readilyremoved from the iodination reaction by centrif gation.It is also possible to re-use the solid state enzyme (20).lodination can be performed at 4°C with high efficiency,a factor which may be advantageous in the case of enzy-mes that are unstable at room temperature.

The results presented here show that α-amylase labelledby Sepharose bound lactoperoxidase, behaved in asimilar manner to unlabelled enzyme in both in vitro andin vivo studies. With the necessary care, the methodshould prove useful for other enzymes which containaccessible tyrosine residues, provided the latter are notinvolved in catalysis.

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Z. Klin, Chen* Klin. Biochem. / 12. Jahrg. 1974 / Heft 12 42A*