Relationship between the medial cuneiform bone morphology and the severity of hallux valgus.

BACKGROUND: It has been reported on the relationship between the medial cuneiform bone morphology, especially in terms of obliquity, and the severity of hallux valgus (HV), however, no consensus has been obtained. On the other hand, there are no reports on the relationship between the difference in height between the medial and intermediate cuneiforms and the severity of hallux valgus. The purpose of this study was to clarify the relationship between the medial cuneiform bone morphology and the severity of HV. METHODS: The authors retrospectively analyzed 200 feet of 116 patients who had a weightbearing anteroposterior foot radiograph taken between April 2017 and July 2022 and diagnosed with HV. Measurements included the hallux valgus angle (HVA), the intermetatarsal angle (IMA), the distal medial cuneiform angle (DMCA) and the cuneiform first-second length (C1-2D). HV groups were classified into one of three groups: mild (15 ≦ HVA<30°, 9 < IMA<13°), moderate (30 ≦ HVA<40°, 13 ≦ IMA≦20°) and severe groups (HVA≧40°, IMA>20°), and the relationship to DMCA or the difference in height between the medial and intermediate cuneiforms was analyzed. RESULTS: A total of 163 feet of 93 patients were included in this study. There were significant correlations between the HVA and the DMCA (r = 0.47, p <0.001) or the C1-2D (r = 0.64, p <0.001). There was no significant difference in DMCA between the mild and moderate groups (p = 0.14). On the other hand, significant differences in C1-2D were observed between the three groups (mild-moderate; p <0.001, moderate-severe; p = 0.03, mild-severe; p <0.001). IMA was also positively correlated with the DMCA (r = 0.30, p <0.001) or the C1-2D (r = 0.47, p <0.001). However, the DMCA (p = 0.07) and the C1-2D (p = 0.39) did not differ significantly from IMA between the moderate and severe groups. CONCLUSIONS: The difference in height between the medial and intermediate cuneiforms, referred to as C1-2D, is closely associated with the HVA. The C1-2D may influence the progression of HV and be a novel radiographic parameter that indicates severity of HV.

Lateral process fracture of the talus in a basketball player treated with arthroscopic reduction-internal fixation: a case report.

An open reduction-internal fixation is generally recommended in Type I lateral process fractures of the talus when the dislocation of the fragment is >2 mm. This report describes a case of a lateral process fracture of the talus in a 24-year-old male basketball player who underwent arthroscopic reduction-internal fixation. The patient complained of pain on the lateral aspect of his left ankle during a cutting motion. Based on physical examination and radiological findings, the patient was diagnosed with a lateral process fracture of the talus and underwent surgical treatment with arthroscopic reduction-internal fixation. At 12 weeks after the surgery, bone union was achieved, and the patient was able to resume playing basketball at his pre-injury level without symptoms, complications, or functional impairment. Lateral process fracture of the talus in a 24-year-old male basketball player was successfully treated with arthroscopic reduction-internal fixation.

Quantification of serine residue stereoinversion in a short peptide by reversed-phase high-performance liquid chromatography: analysis of mechanisms promoting serine stereoinversion.

Stereoinversion of Ser residues within proteins, which has been identified in long-lived proteins, influences protein function. To quantify the stereoinversion of Ser residues, we investigated the potential adaptation of our direct peptide analytical method originally established for analyzing the isomerization of asparaginyl/aspartyl residues. Peptide pairs containing L-Ser or D-Ser residues with lengths of four or five residues were synthesized. Separation conditions for these peptide pairs were systematically examined by precisely adjusting the pH of the elution solvent using reverse-phase high-performance liquid chromatography (HPLC). Optimal separation conditions were successfully developed for all peptide pairs, enabling the direct quantification of Ser residue stereoinversion through a single HPLC run. Subsequently, the degree of Ser stereoinversion within the model peptide, Gly-Ser-Gly-Tyr, was determined using the method established in this study. Surprisingly, the stereoinversion of Ser residues occurred only when the absolute configurations of Ser and Tyr residues of the peptide differed from each other, whereas no stereoinversion was observed when their absolute configurations were identical. The experiments using peptides similar to the model peptide reveal that both the N-terminal amino group and the hydroxyl group of the C-terminal Tyr residue are involved in the stereoinversion of the Ser residue. By applying a simple method to quantify the stereoinversion of Ser residues, valuable insights into the mechanisms governing these stereoinversions were obtained.

Bacillus Calmette-Guérin osteomyelitis of the fifth metatarsal bone in a Japanese infant: a case report.

Bacillus Calmette-Guérin osteomyelitis is a rare complication following Bacillus Calmette-Guérin vaccination. Here, we describe a rare case of Bacillus Calmette-Guérin osteomyelitis of the fifth metatarsal in a 21-month-old Japanese infant. A 21-month-old Japanese female infant presented with a swollen mass on the dorsolateral aspect of the left foot. Based on physical examination, radiological and histopathologic findings and laboratory results, a diagnosis of Bacillus Calmette-Guérin osteomyelitis of the fifth metatarsal bone was made, and an oral anti-tuberculosis treatment was initiated. However, the mass recurred 10 months after the start of the anti-tuberculosis treatment, so additional surgical debridement was performed. Six months after surgery, clinical findings and plain radiograph images revealed complete improvement of the affected area, and anti-tuberculosis treatment was stopped. Bacillus Calmette-Guérin osteomyelitis of the fifth metatarsal in a 21-month-old Japanese infant was successfully treated with oral anti-tuberculosis therapy and surgical debridement.

Chronic Achilles tendon rupture in elderly treated with a combination of the side-locking loop suture technique and early rehabilitation protocol: two cases report.

A chronic Achilles tendon rupture (ATR) is generally defined as a rupture that occurs more than 4-6 weeks after the initial injury. A variety of corrective techniques have been reported, such as direct repair, V-Y plasty, turndown flap, tendon transfer and free tendon grafting. These procedures generally produce good results, but have the disadvantage of requiring prolonged immobilization and weight-bearing restrictions. This may be a risk factor for falls and decreased function in the lower limbs, especially in older patients. Side-locking loop sutures (SLLS) were first introduced in 2010 as a direct repair technique for acute ATR. This technique provides higher tensile strength, which may allow for early rehabilitation protocols such as early range of motion and early weight-bearing of the ankle without postoperative immobilization. In this report, we describe two cases of chronic ATR in elderly patients treated with SLLS and an early rehabilitation protocol.

Fragility fracture of talar neck in an osteoporotic patient treated by posterior-to-anterior screw fixation under hindfoot endoscopy: a case report.

Fragility fractures of the talar neck are extremely rare. Here, we describe a case of fragility fracture of the talar neck associated with osteoporosis in a 76-year-old female, who was treated by posterior-to-anterior screw fixation under hindfoot endoscopy. A 76-year-old female cleaner with a history of osteoporosis complained of pain in her right ankle when going downstairs. Radiological findings revealed a fragility fracture of the talar neck associated with osteoporosis. Because the patient was elderly and it was difficult to treat using a prolonged non-weight-bearing cast, we performed a posterior-to-anterior parallel dual screw fixation under hindfoot endoscopy for this case. As a result, the patient was able to return to work 8 weeks after surgery without pain, dysfunction or complication. Osteosynthesis with posterior-to-anterior screw fixation under hindfoot endoscopy successfully treated a rare case of fragility fracture of the talar neck in a 76-year-old female cleaner.

Preoperative Ahlbäck radiographic classification grade significantly influences clinical outcomes of double level osteotomy for osteoarthritic knees with severe varus deformity.

PURPOSE: The purpose of this study was to examine the relationship between preoperative Ahlbäck radiographic classification grade and the clinical outcomes of double level osteotomy (DLO) performed for osteoarthritic knees with severe varus deformity. METHODS: The study population comprised a consecutive series of 99 knees (68 patients) for which DLO was performed and follow-up results for a minimum of two years were available. The Ahlbäck radiographic classification system was used to determine the osteoarthritic grade. The following radiological parameters for alignment and bone geometry were measured: mechanical lateral distal femoral angle (mLDFA), mechanical medial proximal tibial angle (mMPTA), joint-line convergence angle (JLCA), and mechanical tibiofemoral angle (mTFA). Clinical results were assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and the International Knee Documentation Committee (IKDC) subjective score preoperatively and at 2 years after surgery. Difference between preoperative and postoperative measurements as well as relationship between Ahlbäck grade and radiological/clinical results were statistically assessed. RESULTS: The average age of the study participants was 60.9 ± 6.2 years and the mean follow-up period was 45.4 ± 15.2 months. Each of the radiological parameters exhibited preoperative abnormal values. Knees with Ahlbäck grade 3 and 4 osteoarthritis exhibited significantly greater JLCA and mTFA than grade 1 knees. Two years post-surgery, all radiological parameter values measured within a normal range. Clinical evaluation showed significant improvement in KOOS after surgery. Analysis of the relationship between Ahlbäck grade and clinical score showed that the 2-year postoperative KOOS scores in grade 3 and 4 osteoarthritic knees were significantly lower than grade 1 knees (with the mean 2-year KOOS scores of 350.0 ± 79.9, 317.9 ± 78.3, and 420.2 ± 42.9, respectively). CONCLUSIONS: While DLO may produce significant radiological and clinical improvement in knees with joint space obliteration, Ahlbäck grade 3 and 4 osteoarthritic knees associated with larger JLCA and mTFA showed less satisfactory clinical results compared to grade 1 knees. LEVEL OF EVIDENCE: IV case series.

Talar Body Fracture Treated by Hindfoot Endoscopic Reduction and Internal Fixation.

Background: A talar body fracture is relatively rare. Surgical treatment for the fracture is generally indicated for the displaced fracture types and traditionally performed via open approaches. However, open approaches may not be able to achieve adequate exposure of the talus body owing to the difficulty in achieving reduction and fixation of the fracture. In this case report, we describe a case of talar body fracture treated by hindfoot endoscopic reduction and internal fixation. Case report. A 39-year-old man who was a carpenter complained of right hindfoot pain after he fell from a stepladder during work. Although plain radiographs of the right ankle showed no abnormal findings, noncontrast computed tomography demonstrated a fracture line from the lateral side of the posterior lateral process to the medial side of the talus body. In addition, the fracture line extended to the posterior ankle and subtalar joints, and the bone fragment was slightly displaced. We diagnosed him with the talar body fracture and performed operative treatments using hindfoot endoscopic reduction and internal fixation. At 14 weeks after the operation, he was able to return to work at the preinjury activity level without dysfunction of the ankle nor complications. Conclusion: We reported a case of talar body fracture treated by hindfoot endoscopic reduction and internal fixation. In this case, the hindfoot endoscopic technique provided visualization of the fracture site with less invasiveness and increased safety, which enabled proper reduction and internal fixation of the fracture site. Therefore, the patient was able to return to work at the preinjury activity level at 14 weeks after surgery without dysfunction of the ankle nor complications. This surgical technique may be a useful option for the talar body fracture.

The Effectiveness of Intramedullary Screw Fixation Using the Herbert Screw for Fifth Metatarsal Stress Fractures in High-Level Athletes.

BACKGROUND: Intramedullary screw fixation is the most common operative procedure used for treatment of fifth metatarsal stress fractures in athletes. However, the optimal implant in intramedullary screw fixation is still being investigated. PURPOSE: To review experiences with intramedullary screw fixation using the Herbert screw for fifth metatarsal stress fractures in high-level athletes. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: The authors retrospectively analyzed 37 high-level athletes (Tegner activity score ≥7) who underwent intramedullary screw fixation using the Herbert screw for fifth metatarsal stress fractures between August 2005 and August 2017. The minimum follow-up period of the patients was 2 years. In assessing the surgical results, time to obtain bone union, time to return to original level of sport participation, and treatment failures/complications were reviewed. Additionally, the effect of intraoperative plantar gap widening caused by the screw insertion was analyzed. The surgical results of the 2 groups, the no-gap group (intraoperative plantar gap widening, <1 mm) and the gap group (intraoperative plantar gap widening, ≥1 mm), were compared, while correlations between intraoperative plantar gap widening and the surgical results were statistically analyzed. RESULTS: Bone union and return to the original sport were attained in all patients without treatment failures/complications such as delayed union, nonunion, or refracture. The mean time to obtain bone union was 10.1 weeks, and the mean time to return to sport was 10.9 weeks. In comparing the no-gap group (n = 16) and the gap group (n = 21), no significant differences in the time to obtain bone union (P = .392) or to return to sport (P = .399) were noted. Additionally, there was no correlation between intraoperative plantar gap widening and the time to obtain bone union (r = 0.131; P = .428) or to return to sport (r = 0.160; P = .331). CONCLUSION: The use of the Herbert screw for intramedullary screw fixation to treat fifth metatarsal stress fractures in high-level athletes provided satisfactory results enabling all the athletes to return to the original sport without treatment failures/complications. Additionally, intraoperative plantar gap widening does not affect the surgical results using this technique.

Return to the original sport at only 3 months after an Achilles tendon rupture by a combination of intra-tissue injection of freeze-dried platelet-derived factor concentrate and excessively early rehabilitation after operative treatment in a male basketball player: A case report.

BACKGROUND: Achilles tendon rupture is one of the most common serious injuries in athletes. Various studies to accelerate the healing process of the Achilles tendon have been performed as it takes a longer time to repair the tissue compared to other tendons. Here, we report a case of an acute Achilles tendon rupture in a male basketball player treated by a combination of an intra-tissue injection of freeze-dried platelet-derived factor concentrate, which included a platelet-derived growth factor with an early rehabilitation protocol after the operative treatment to facilitate the biological healing of the injured tendon tissue. To the best of our knowledge, this case is the first instance that enabled the athlete to return to original sport activity at only 3-months after the injury. CASE REPORT: A 23-year-old male basketball player who belonged to a university basketball team sustained an Achilles tendon rupture during running in a training match. The remaining time period until the final tournament of the university league as a senior player was only 3 months. The patient received a combination of an intra-tissue injection of freeze-dried platelet-derived factor concentrate and early rehabilitation protocol after operative treatment. Surgery was performed 4 days after the injury and the early rehabilitation protocols were applied postoperatively. A freeze-dried platelet-derived factor concentrate was injected into the ruptured site of the Achilles tendon under ultrasound guide at 4 weeks postoperatively. The patient could return to play at the pre-injury level without any symptoms and disfunctions at 3 months after surgery. At two years postoperatively, the patient could play basketball without symptoms or rerupture. CONCLUSIONS: We reported a case of an Achilles tendon rupture which was treated by a combination of intra-tissue injection of freeze-dried platelet-derived factor concentrate and an early rehabilitation protocol after the operative treatment. The patient could return to play basketball at the pre-injury activity level at only 3-months after the injury, suggesting that the role of applying excessively early rehabilitation of mechanical loading could facilitate tendon tissue healing when combined with an intra-tissue injection of freeze-dried platelet-derived factor concentrate.

Effect of amino acids present at the carboxyl end of succinimidyl residue on the rate constants for succinimidyl hydrolysis in small peptides.

Structural alterations of aspartyl and asparaginyl residues in various proteins can lead to their malfunction, which may result in severe health disorders. The formation and hydrolysis of succinimidyl intermediates are crucial in specific protein modifications. Nonetheless, only few studies investigating the hydrolysis of succinimidyl intermediates have been published. In this study, we established a method to prepare peptides bearing succinimidyl residues using recombinant protein l-isoaspartyl methyltransferase and ultrafiltration units. Using succinimidyl peptides, we examined the effect of amino acid residues on succinimidyl hydrolysis at the carboxyl end of succinimidyl residues and determined the rate constant of hydrolysis for each peptide. The rate constant of succinimidyl hydrolysis in the peptide bearing a Ser residue at the carboxyl side (0.50 ± 0.02 /h) was 3.0 times higher than that for the peptide bearing an Ala residue (0.17 ± 0.01 /h), whereas it was just 1.2 times higher for the peptide bearing a Gly residue (0.20 ± 0.01 /h). The rate constant of succinimidyl formation in the peptide bearing a Ser residue [(2.44 ± 0.11) × 10-3 /d] was only 1.2 times higher than that for the peptide bearing an Ala residue ([1.87 ± 0.09) × 10-3 /d], whereas 5.5 times higher for the peptide bearing a Gly residue [(10.2 ± 0.2) × 10-3 /d]. These results show that the Gly and Ser residues at the carboxyl end of the succinimidyl residue have opposing roles in succinimidyl formation and hydrolysis. Catalysis of Ser residue's hydroxyl group plays a crucial role in succinimidyl hydrolysis.

Nonunion of a Stress Fracture at the Base of the Second Metatarsal in a Soccer Player Treated by Osteosynthesis with the Bridging Plate Fixation Technique.

BACKGROUND: A stress fracture of the second metatarsal base in soccer players is extremely rare. In this case study, we report a nonunion of a stress fracture at the base of the second metatarsal in a female soccer player who had persistent pain despite continued conservative treatment, who then was treated with the bridging plate fixation technique. Case Report. A 19-year-old female college soccer player complained of pain on the dorsum of her right midfoot during a game without history of trauma and was conservatively treated for 6 months. Radiographic examination showed an oblique fracture with small bone fragment at the proximal base of the second metatarsal and computed tomography demonstrated sclerotic change around the fracture site. We diagnosed her with nonunion of a stress fracture at the base of the second metatarsal and performed operative treatments using autogenous cancellous iliac bone grafting and plate fixation bridging a second metatarsal and medial cuneiform with a locking plate. At 4 months after the initial surgery, she was able to return to playing soccer at preinjury level without complications or pain. CONCLUSION: We report a rare case of nonunion of a stress fracture at the base of the second metatarsal in a female soccer player without underlying diseases. Surgical treatment was applied, because the conservative treatment was ineffective for 6 months and led to nonunion. The plate fixation technique bridging the second metatarsal and medial cuneiform was a useful option to attain the bone fusion for small fracture fragment for a treatment of nonunion of a stress fracture at the base of the second metatarsal.

Laparoscopic transhiatal suture closure for spontaneous esophageal rupture: a case report.

BACKGROUND: Spontaneous esophageal rupture is a rare but serious disease with high mortality. Conservative treatment and endoscopic therapy have been reported, but surgical treatment is still a basic modality of therapy. In addition to thoracotomy, recent studies have reported treatment with thoracoscopic surgery and laparoscopic transhiatal repair. In this study, we report a patient who underwent laparoscopic transhiatal suture closure for spontaneous esophageal rupture with favorable postoperative course. We also discuss indication for laparoscopic surgery for spontaneous esophageal rupture. CASE PRESENTATION: A 70-year-old man visited our hospital with chief complaints of epigastric pain and vomitus niger. He was diagnosed with spontaneous esophageal rupture in the left wall of the lower esophagus by computed tomography and upper gastrointestinal (GI) series. At 11 h after the onset of symptoms, we performed laparoscopic transhiatal suture closure and lavage drainage. We performed transhiatal esophageal replacement using the 5-hole approach. We observed a perforation of 2 cm in diameter at the site of the rostral portion approximately 4 cm from the esophageal hiatus. All layers were closed with three stitches using 3-0 absorbable sutures. No perforation was observed in the thoracic cavity. The total operative time was 178 min, and total bleeding was 2 ml. He had no postoperative complications and was discharged on day 15 after the procedure. He received continuous proton pump inhibitor therapy as an outpatient. Healing cicatrization was found at the site of rupture by esophagogastroscopy. The patient was advised to improve his lifestyle and has shown no signs of recurrence over 2 years from the date of surgery. CONCLUSIONS: Simple closure of all the layers using laparoscopic transhiatal simple closure was useful in the treatment of esophageal rupture as a less invasive approach for patients who meet the following conditions: stable general condition, intrathoracic perforation, and the perforation site is identified as the lower esophagus by pre-operative examination.

Tag-Convertible Photocrosslinker with Click-On/Off N-Acylsulfonamide Linkage for Protein Identification.

The N-acylsulfonamide group, known as a safety-catch linker, has been applied to photoaffinity labeling (PAL) using a cinnamate-type photocrosslinker to improve the efficiency of PAL-based target identification. A bioorthogonal sulfo-click reaction was used to stably link a photocrosslinker unit with N-acylsulfonamide linkage to produce a photoactivatable probe without any protection. In addition, the crosslinked protein was selectively isolated with a small cinnamate tag via linkage disruption upon N-alkylation. Furthermore, the tag moiety was photochemically converted to a stable coumarin derivative by losing a water molecule, which is a useful property in MS-based identification.

Photoinduced Electron Transfer-Regulated Protein Labeling With a Coumarin-Based Multifunctional Photocrosslinker.

We developed a novel diazirine-based photolabeling agent having a (coumarin-4-yl)methyl ester scaffold, which exhibited multiple photochemical properties of crosslinking, fluorogenicity and cleavage. These properties can be kinetically regulated via photoinduced electron transfer between diazirine and coumarin moieties. The C-O bond of (coumarin-4-yl)methyl ester can be cleaved via photochemical excitation of coumarin moiety, that function has been initially quenched by the diazirine moiety. Upon diazirine photolysis with 365-nm light, interacting protein was stably captured with photoactivatable ligand probe. Then, the unlocked cleavage function was activated with 313 nm light, and the reaction was accelerated in a weakly-basic solution. The crosslinked protein could be selectively isolated with attachment of a small coumarin tag on the surface. This multi-functional labeling agent has a great potential to facilitate LC-MS/MS-based protein identification.

Induced membrane technique using beta-tricalcium phosphate for reconstruction of femoral and tibial segmental bone loss due to infection: technical tips and preliminary clinical results.

PURPOSE: This study aimed to provide preliminary evidence regarding effectiveness of grafting beta-tricalcium phosphate (ß-TCP) combined with a cancellous autograft for treating nonunion of long bones in the lower extremity due to infection by evaluating clinical and radiological outcomes. METHODS: We retrospectively reviewed the clinical and radiological results in seven patients (six men, one woman; median age 39 years) treated by the induced membrane technique for nonunion of the femur or tibia due to infection. In the second stage of the procedure, the bony defect was filled with a combination of autologous cancellous bone and ß-TCP, which were mixed in approximately the same proportions. The time interval between the second stage of the procedure and bone healing was investigated. Radiographic characteristics including maximum bone gap and radiographic apparent bone gap were evaluated. RESULTS: The median follow-up period was 14 months. Bone healing was achieved in a median of six months after the second procedure. The median maximum bone gap and radiographic apparent bone gap were 55 mm and 34 mm, respectively. DISCUSSION: Use of ß-TCP, which has osteoconductive ability, with an autograft provided good clinical and radiological outcomes. The findings of this preliminary study suggest the potential of ß-TCP as a useful bone substitute for autografts in the induced membrane technique. CONCLUSIONS: Our findings suggest that ß-TCP may be an effective extender when using the induced membrane technique.

Kinetic controlled affinity labeling of target enzyme with thioester chemistry.

High specificity has been an important feature in affinity labeling for target profiling. Especially, to label targets via rapidly progressing reactions with consumption of ligand (probe), high specificity of reaction with common functional groups of target protein should be achieved without reactions with similar groups of non-target proteins. Herein, we demonstrate the kinetic controlled affinity labeling of acyl CoA synthetase using a fatty acid analogue containing a phenylthioester linkage. High specificity was attained by accelerating the labeling rate in the binding pocket. This approach could be useful for profiling a series of target enzymes and transporters in signal transduction pathways.

Relationship between Virucidal Efficacy and Free Iodine Concentration of Povidone-Iodine in Buffer Solution.

Povidone-iodine solutions prepared to various concentrations (0.01, 0.1, 1 and 10%) with 0.2M phosphate buffer (pH 7.0) (PVP-I PB) were analyzed to determine their free iodine concentrations using membrane permeation cells, and their inactivation effects on three viruses (influenza A virus, poliovirus type 1 and adenovirus type 3) were examined. The free iodine concentrations in the 0.01-10% PVP-I PB were determined to be 1.84, 4.88, 1.58 and 0.17 ppm (approximate values), respectively, with the maximum obtained for the 0.1% solution. The virucidal efficacy of these PVP-I PB against poliovirus type 1 and adenovirus type 3 was found to be generally dependent on free iodine concentration, with the 0.1% solution being the most effective. Influenza A virus was inactivated with an action time of 15 s at all four concentrations examined. The results of this study suggested an association between free iodine concentration and virucidal efficacy for the 0.01-10% PVP-I PB.

Structure-assisted ligand-binding analysis using fluorogenic photoaffinity labeling.

Photoaffinity labeling (PAL) technique using a fluorogenic cross-linker is used to monitor the nucleotide-binding pocket within a protein. A coumarin fluorophore formed in the binding domain due to ultraviolet (UV) irradiation has been shown to accelerate the sequencing of the labeled peptide as well as identification of the labeled site by liquid chromatography (LC)-tandem mass spectrometry (MS), in addition to providing information on the ligand binding state. Selective monitoring of the predefined fluorescence peaks among the numerous digests obtained from high performance liquid chromatography (HPLC) clearly indicates the binding capability of the ligand to the entire protein as well as to the corresponding binding domain under various conditions. In the current study, ligand-binding analysis confirmed by the structural information of the binding state has been demonstrated using fluorogenic ATP/ADP photoactivatable probes under allosteric regulation of multiple substrates in the enzyme glutamate dehydrogenase (GDH).