Referring to a traditional Chinese methodology, illness of patient can be detected bysilver, usually in form of coin or strip inside a hard-boiled egg. The silver strip, whenrolled with albumen on the area where patient felt sick, shows obvious colour changesfrom silvery white to yellow, red, blue or purple, depending on the type of illness.While the correlation between the colour changes and the nature of the illness hadremained uncertain, we found linkages between the methodology and silver stainingfor protein footprinting, which was introduced by Switzer et al. in 1979.1 First of all,the colour changes matched with what reported by Chao-Ming Tsai and Carl E.Frasch.2 Secondly, the rate of colour change had increased as the reactionprogressed.3 Furthermore, both colour changes involve the interaction between silverand protein, which is abundant in egg. Hence, we suggested that the classic Chinesemethodology might have a mechanism similar to that of colour silver staining. Severalexperiments were carried out to replicate the methodology, with different externalfactors being investigated.
The Mechanistic Study on Traditional Chinese Silver Staining and
its Application as Phosphate Ion Detection
Mechanism of Chinese Silver Staining Diagnosis and its
Application for Phosphate Ion DetectionKings CollegeChu Yat HoYip
Tsz FungDavid IuZuo Long LongHo Hiu Fai
Traditional Chinese Silver Staining Technique
(1) INTRODUCTIONWhy does silvers colour change?HypothesisAg
undergoes specific reaction with the proteins and amino acids
present in the egg white.
ExperimentationTest different amino acids
suspectedLeucineMethionineCysteinePhenylalanineAsparagineHistidineAlanineProlineGlutamic
acidGlycine
ValineAspartic acid(2) MECHANISMResult: Only cysteine show
characteristic color change
Purple and blue(2) MECHANISM
Silvery whiteResult: Only cysteine show characteristic color
changepH dependenceConditionColour changeResultAcidicSilver to
YellowAlkalineSilver to Blue
Question 2: Why color depends on pH?Hypothesis: Cysteine has
different structure under different pH conditions.What is
CYSTEINE?
It has unique thiol group, which we proposed is responsible for
coloration.pH dependence
Proposed mechanism
DiscussionCarboxylate group is responsible for the yellow
complexes in acidic conditionAmine group is responsible for the
blue complexes in alkaline conditionBlue complexes are under
thermodynamic control
VerificationHypothesis: Cysteamine will give yellow complexes,
3-mercaptopropinoic acid will give blue complexes, irrespective to
pH conditions.
Experimentation: use 3-mercaptopropinoic acid and cysteamine to
verify the mechanism.
(Carboxyl group removed)(Amine group
removed)CysteineCysteamine3-Mercaptopropionic acidpH 4.0
bufferYellowNo observable changeYellowpH 10.0
bufferBlueBlueYellow
Catalytic Effect of anionsExperimentation: to test the catalytic
effect of different anions on the coloration on silver plate
Results:PO43- HCO3- Cl-PO43- +Cl- + HCO3-PO43-HCO3-Cl-
Colour of the silver plateControl (Distilled water was
added)(Overall conc. of PO43- : 0 M )2.0 cm3 5.00 x 10-6 M PO43-
added(Overall conc. of PO43- : 3.125 x 10-7 M )2.0 cm3 5.00 x 10-5
M PO43- added(Overall conc. of PO43- : 3.125 x 10-6 M ) 2.0 cm3
5.00 x 10-4 M PO43- added(Overall conc. of PO43- : 3.125 x 10-5 M
)
DiscussionPhosphate ion can significantly promote the coloration
in both acidic and alkaline buffer solution.
The coloration speeds up from 1 hr to 10 min under the presence
of phosphate ion.Screening test for Chronic Renal FailureChronic
Renal Failure patients exhibit proteinuriaProtein concentration in
urine >0.43 g/dm3High level of proteins & amino acids
discharged in urine Measurement of protein concentration = test for
proteinuriaScreening test for Chronic Renal FailureSuggested
procedureCollect first morning urine samples from test
subjectsRefrigerated at 4oC for
