A protein biosensor for lactate.

Blood lactate is a clinically valuable diagnostic indicator. In this preliminary report we describe a protein biosensor for L-lactate based on beef heart lactate dehydrogenase (LDH). LDH was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled LDH displayed an approximately 40% decrease in emission intensity upon binding lactate. This decrease can be used to measure the lactate concentration. The ANS-labeled LDH was further utilized in a new sensing format, polarization sensing, which is suitable for miniaturization to a point-of-care lactate monitor. However, temporal instability of beef heart LDH indicates the need for further protein engineering prior to development of a more robust lactate-sensing protein.

[1]  M. P. Platt,et al.  Association between blood lactate and acid-base status and mortality in ventilated babies , 1997, Archives of disease in childhood. Fetal and neonatal edition.

[2]  R. Schneiter,et al.  Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, XI. Engineering thermostability and activity of lactate dehydrogenases from bacilli. , 1991, Biological chemistry Hoppe-Seyler.

[3]  P Argos,et al.  Engineering protein thermal stability. Sequence statistics point to residue substitutions in alpha-helices. , 1989, Journal of molecular biology.

[4]  J. Lakowicz,et al.  Polarization sensing with visual detection. , 1999, Analytical chemistry.

[5]  I Gryczynski,et al.  Glucose sensor for low-cost lifetime-based sensing using a genetically engineered protein. , 1999, Analytical biochemistry.

[6]  W. Cascio,et al.  A conducting salt-based amperometric biosensor for measurement of extracellular lactate accumulation in ischemic myocardium. , 1997, Analytical chemistry.

[7]  J. Lakowicz,et al.  Simple apparatus for polarization sensing of analytes , 2000 .

[8]  T. Clerbaux,et al.  Assessment of the lactate biosensor methodology. , 1998, The European respiratory journal.

[9]  J. Vincent,et al.  Serial blood lactate levels can predict the development of multiple organ failure following septic shock. , 1996, American journal of surgery.

[10]  Catalano,et al.  Room temperature lasing at blue wavelengths in gallium nitride microcavities , 1999, Science.

[11]  B Persson,et al.  Lactate compared with pH analysis at fetal scalp blood sampling: a prospective randomised study , 1998, British journal of obstetrics and gynaecology.

[12]  Henryk Szmacinski,et al.  Micro- and Sub-Nanosecond Lifetime Measurements Using a UV Light-Emitting Diode , 2000 .

[13]  J R Lakowicz,et al.  A lifetime-based optical CO2 gas sensor with blue or red excitation and stokes or anti-stokes detection. , 1995, Analytical biochemistry.

[14]  A. Rocchini,et al.  Lactate blood levels in the perioperative period of orthotopic liver transplantation , 1997, International journal of clinical & laboratory research.

[15]  Joseph R. Lakowicz,et al.  Single quantum well light emitting diodes demonstrated as excitation sources for nanosecond phase‐modulation fluorescence lifetime measurements , 1996 .

[16]  J. Castillo,et al.  Intrinsic molecular fluorescence of lactate dehydrogenase: an analytical alternative for enzymic determination of pyruvate. , 1997, The Analyst.

[17]  J. Lakowicz,et al.  Anisotropy-based sensing with reference fluorophores. , 1999, Analytical biochemistry.

[18]  C. Burtis.  Tietz textbook of Clinical Chemistry , 1994 .

[19]  V. V. Mozhaev,et al.  Structure-stability relationships in proteins: a guide to approaches to stabilizing enzymes , 1990 .

[20]  J. Lakowicz,et al.  The β-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: enzyme activity and conformational dynamics at temperatures above 100°C , 1999 .

[21]  Joseph R. Lakowicz,et al.  Blue light-emitting diode demonstrated as an ultraviolet excitation source for nanosecond phase-modulation fluorescence lifetime measurements , 1997 .

[22]  M. Ivanov,et al.  The use of auramine O to study ligand binding and subunit cooperativity of lactate dehydrogenase. , 1984, Biochimica et biophysica acta.

[23]  Joseph R. Lakowicz,et al.  A 10‐GHz frequency‐domain fluorometer , 1990 .

[24]  Jan D. Miller,et al.  Anomalous Dispersion Effects in the IR-ATR Spectroscopy of Water , 2000 .

[25]  E Gratton,et al.  Quantitative determination of the absorption spectra of chromophores in strongly scattering media: a light-emitting-diode based technique. , 1994, Applied optics.

[26]  G. Royer,et al.  Substrate-induced conformational changes in lactate dehydrogenase. Proteolysis of the immobilized enzyme in the presence of specific substrates. , 1977, The Journal of biological chemistry.

[27]  D. Centonze,et al.  A microdialysis fibre based sampler for flow injection analysis: determination of L-lactate in biofluids by an electrochemically synthesised bilayer membrane based biosensor. , 1996, Biosensors & bioelectronics.