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ProClin
®
Preservatives
Mechanisms and Stabilityfor Diagnostics Reagents
 
www.safcsupplysolutions.com/proclin
ProClin
®
Preservatives
Mechanisms and Stability
for Diagnostic Reagents
 
ProClin
®
Preservatives
Mechanisms and Stability
The unique biocidal mechanisms of ProClin preservatives provide a broadspectrum of activity, strong lethality at low concentration and preclude microbial
resistance by mutation. In addition, the biocidal isothiazolone components
in these preservatives offer a wide range of chemical compatibility and pHtolerance. Combined, these characteristics make ProClin preservatives an idealchoice for a variety of diagnostic reagent systems.
The Mechanism of Action of ProClinPreservatives
ProClin preservatives are immediatelybacteriostatic upon contact with a microbe(Figure A). This is the result of the ability of the active ingredients
1
to quickly penetrate
cell membranes and inhibit specicenzymes in the cell. Some of these targetenzymes are within the central metabolic
cycle of the cell, the Krebs cycle.ProClin preservatives attack the Krebs
cycle at four sites: the enzymes pyruvatedehydrogenase, α-ketoglutarate
dehydrogenase, succinate dehydrogenase,and NADH dehydrogenase (Figure B). Withthe Krebs cycle debilitated, cells rapidlylose the ability to produce energy andsubsequently die.Because all bacteria and fungi possessat least part of the Krebs cycle, ProClinpreservatives are broad spectrum in theiractivity. The ability of ProClin preservatives
to act on specic enzymes is reected in
the low levels required to control growth.ProClin preservatives also target multiple
specic enzymes, reducing the microbes
ability to mutate one target site to achieveany level of resistance.The rapid disruption of cellular metabolism
as a result of specic enzyme inhibition
severely impairs the ability of the cell
to repair damage inicted upon its
components. The accumulation of damagebeyond the capacity of the cell for repairresults in cell death. Low concentrationsof ProClin preservative, which areimmediately bacteriostatic, requireseveral hours to kill the cell, while higherconcentrations exhibit rapid microbicidal
effects (Figure C). This is a reection of the
rate-driven nature of the damage process;i.e. higher concentrations of ProClin
preservative both inict damage at a
greater rate and overwhelm the cell’s repairfunctions faster than lower concentrationsof ProClin preservative.
1ppm
Hours After Biocide Addition
   A   b  s  o  r   b  a  n  c  e  a   t   6   0   0  n  m
0.2-0.50.0 0.51.0 1.5
ProClin ActiveIngredient Level
0.00.50.40.30.10ppm2.5ppm
Figure A.Rapid Inhibition of Growth
GlucosePyruvate2H CO
2
 Acetyl-CoAOxaloacetateCitrate[
cis 
-Aconitate]Isocitrate
α
-KetogluatrateSuccinyl-CoASuccinateFumarateMalateNADElectron Transport ChainKREBSCYCLECO
2
CO
2
2H 2H2H2HSites of ProClin biocide inhibition
Figure B.Sites of Inhibition
(1) 
5-chloro-2-methyl-4-isothiazolin-3-one (MCI, CMIT, RH-651) and 2-methyl-4-isothiazolin-3-one (MI, MIT, RH-573)
www.safcsupplysolutions.com/proclin
 
for Diagnostic Reagents
Data indicates that ProClin preservativespossess multiple pathways that result inthe lethal loss of protein thiols (Figure D):
Covalent modication via direct
1.electrophilic attackGeneration of a secondary2.
electrophile by disulde exchange andtautomerization to a thioacyl chloride
 An intracellular generation of free3.radicals as a result of the severemetabolic disruption, which severelystresses the cell’s natural radicaldefense mechanism
Stability of ProClin Preservatives inAmino Buffers
ProClin preservatives exhibit a widerange of chemical compatibility and pHtolerance, making them an ideal choicefor preserving a variety of systems. Oneclass of chemicals that is aggressive to
the isothiazolone molecules is secondary
amines. With their strong nucleophilicactivity, secondary amines break the
isothiazolones’ sulfur-nitrogen bond,
resulting in ring-opening and loss of biocidal activity. Much of this interactionis pH-dependent; at alkaline pH theequilibrium favors amines that are highlyaggressive nucleophiles. At neutral pH andbelow, the amines are protonated and havereduced nucleophilic activity.Table 1. The Effect of pH on Stability
Buffer(50mM)pH% Loss ofActive ProClinPreservative After4 weeks at 25 °C% Loss ofActive ProClinPreservative After4 weeks at 40 °C
TRIS 7.0 6.3 4.8TRIS 8.0 20.3 36.8HEPES 7.0 8.3 9.8HEPES 8.0 12.0 45.0TES 7.0 8.1 8.2TES 8.0 19.4 39.1
 Amino-based buers are common componentso diagnostic reagents. The stability o ProClin preservatives in various amino buers, and theeect o pH on this stability, have been studied.The results indicate that biocide stability is strongly correlated with buer pH (Table 1). At  pH 7.0, there is a small loss o active biocide ater 4 weeks storage (25°C) in all the buerstested. Degradation accelerated signifcantly when the pH o the buers was 8.0. The results also indicated that amine-mediated degradationoccurred at a much higher rate when the storagetemperature was increased rom 25°C to 40°C.
In order to maximize the duration of 
antimicrobial protection, amino-basedbuffers should be adjusted to pH 7.0 orlower, whenever possible, prior to addingProClin 150, 200 or 300 preservatives.If the pH of the reagent system is criticalto the diagnostic assay, ProClin 950 shouldbe considered, as well as an alternativebuffer system. Buffers based on stericallyhindered tertiary amines may be morecompatible with ProClin preservativesthan those based on primary orsecondary amines.
SRSHOCISCH
3
HSCIONCH
3
SRSSN
Disruption of MetabolismRadical CascadeIrreparable DamageDeath
CIONCH
3
SHNCH
3
OCISSHOHNH
2
RS
MCI
PROTEINPROTEINPROTEINPROTEIN
Figure D.Cells Killed vs. Protein Thiols LostFigure C.Cidal Activity of ProClin Preservative(Cell Death)
10
-2
10
-3
10
-1
10
0
10
1
10
2
   %    S  u  r  v   i  v  o  r  s
06510 45 095703
100ppm MCI25ppm MCI5ppm MCI*
Minutes After Biocide Addition
* MCI = methylchloroisothiazolone (primary active ingredient inProClin preservatives)
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